Novel rsv rna molecules and compositions for vaccination

ABSTRACT

The present invention is directed to an artificial nucleic acid, particularly to an artificial RNA suitable for use in treatment and/or prophylaxis of an infection with Respiratory syncytial virus (RSV) or a disorder related to such an infection. The invention further concerns a method of treating or preventing a disorder or a disease, first and second medical uses of the artificial RNA, compositions, and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the artificial RNA, compositions and vaccines.

INTRODUCTION

The present invention is directed to artificial RNA suitable for use in the treatment or prophylaxis of an infection with Respiratory syncytial virus (RSV) or of a disorder related to such an infection. In particular, the artificial RNA of the invention comprises at least one heterologous untranslated region (UTR), preferably a 3′-UTR and/or a 5′-UTR, and a coding region encoding at least one antigenic peptide or protein derived from RSV, in particular at least one antigenic peptide or protein derived from RSV fusion (F) protein. The artificial RNA is preferably characterized by increased expression efficacies of coding regions operably linked to said UTR elements. The present invention is also directed to compositions and vaccines comprising said artificial RNA in association with a polymeric carrier, a polycationic protein or peptide, or a lipid nanoparticle (LNP). Further, the invention concerns a kit, particularly a kit of parts comprising the artificial RNA or composition or vaccine. The invention is further directed to a method of treating or preventing a disorder or a disease, and first and second medical uses of the artificial RNA, composition, or vaccine.

Respiratory syncytial virus (RSV) is an enveloped non-segmented negative-strand RNA virus in the family Paramyxoviridae, genus Pneumovirus. It is the most common cause of bronchiolitis and pneumonia among children in their first year of life. RSV also causes repeated infections including severe lower respiratory tract disease, which may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. Currently, passive immunization is used to prevent severe illness caused by RSV infection, especially in infants with prematurity, bronchopulmonary dysplasia, or congenital heart disease.

Recommended treatment of RSV bronchiolitis primarily consists of respiratory support and hydration. No specific anti-viral therapy is recommended. The neutralizing monoclonal antibody Palivizumab is used for prophylaxis of infants at highest risk for severe infection but is too expensive and impractical for universal use. Currently, there is no licensed/approved RSV vaccine, and developing a safe and effective RSV vaccine is a global public health priority.

In a vaccine trial in the 1960s, infants and young children were immunized with a formalin-inactivated whole virion RSV preparation (FIRSV) or an equivalent paramyxovirus preparation (FIPIV). Five percent of the subjects who were immunized with F-PIV and then naturally infected by RSV during the next RSV season were hospitalized; 80% of those who were immunized with FI-RSV and then infected by RSV were hospitalized, and two children died. This enhancement of an RSV infection due to vaccination is a specific problem for the development of vaccines against RSV infections.

Therefore, Respiratory syncytial virus (RSV) infections are the greatest remaining unmet infant vaccine need in developed countries and an important unmet infant vaccine need worldwide. More than 40 years of effort have not yet resulted in a licensed RSV vaccine for humans.

Despite the above mentioned humanized monoclonal antibody Palivizumab, live-attenuated vaccine viruses were developed which elicit a strong immune response, but which are not recommended for use in the specific target groups (infants, children, the elderly and immunocompromised patients). Also, DNA vectors expressing RSV F protein which bears B-cell epitopes were used to induce the production of neutralizing antibodies. In this context, WO2008/077527 and WO96/040945 disclose vectors comprising DNA sequences encoding RSV F protein for the use as vaccines. However, the use of DNA as a vaccine may be dangerous due to unwanted insertion into the genome, possibly leading to interruption of functional genes and cancer or the formation of anti-DNA antibodies.

WO2015/024668 discloses RNA sequences encoding RSV antigenic peptides and proteins selected from fusion protein F, the glycoprotein G, the short hydrophobic protein SH, the matrix protein M, the nucleoprotein N, the large polymerase L, the M2-1 protein, the M2-2 protein, the phosphoprotein P, the non-structural protein NS1 or the non-structural protein NS2, and an antigenic composition comprising protamine-complexed RNA suitable for intradermal administration.

WO2017/070622 discloses a vaccine comprising RNA encoding RSV antigenic peptides and proteins selected from glycoprotein F and glycoprotein G, wherein the RNA is formulated in lipid nanoparticles.

Apart from some approaches cited above, there remains an unmet medical need for an efficient vaccine for prophylaxis or treatment of RSV infections.

Accordingly, it is the object of the underlying invention to provide novel artificial RNA coding for antigenic peptides or proteins of RSV and compositions/vaccines comprising said RNA for the use as vaccine for prophylaxis or treatment of RSV infections, particularly in infants, newborns, pregnant women, elderly, and immunocompromised patients.

Further it would be desirable that an RNA-based composition or vaccine has some of the following advantageous features:

-   -   Improved translation of RNA constructs at the site of injection         (e.g. muscle)     -   Very efficient induction of RSV antigen-specific immune         responses against the encoded antigenic peptide or protein at a         very low dosages and dosing regimen.     -   Suitability for maternal immunization     -   Suitability for vaccination of infants and/or newborns     -   Suitability for intramuscular administration     -   Induction of an RSV-specific functional humoral immune response     -   Induction of RSV-specific B-cell memory     -   Faster onset of immune protection against RSV     -   Longevity of the induced immune responses against RSV     -   Induction of broad cellular T-cell responses against RSV     -   Induction of a (local and transient) pro-inflammatory         environment     -   No induction of systemic cytokine or chemokine response after         application of the vaccine     -   Well tolerability, no side-effects, non toxic,     -   No enhancement of an RSV infection due to vaccination     -   Advantageous stability characteristics of the vaccine     -   Speed, adaptability, simplicity and scalability of RSV vaccine         production

The objects outlined above are solved by the claimed subject matter.

Definitions

For the sake of clarity and readability the following definitions are provided. Any technical feature mentioned for these definitions may be read on each and every embodiment of the invention. Additional definitions and explanations may be specifically provided in the context of these embodiments.

Percentages in the context of numbers should be understood as relative to the total number of the respective items. In other cases, and unless the context dictates otherwise, percentages should be understood as percentages by weight (wt.-%).

Adaptive immune response: The term “adaptive immune response” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to an antigen-specific response of the immune system (the adaptive immune system). Antigen specificity allows for the generation of responses that are tailored to specific pathogens or pathogen-infected cells. The ability to mount these tailored responses is usually maintained in the body by “memory cells” (B-cells). In the context of the invention, the antigen is provided by the artificial RNA coding sequence encoding at least one antigenic peptide or protein.

Antigen: The term “antigen” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells. Also fragments, variants and derivatives of peptides or proteins derived from e.g. RSV F protein comprising at least one epitope are understood as antigens in the context of the invention. In the context of the present invention, an antigen may be the product of translation of a provided artificial RNA as specified herein.

Antigenic peptide or protein: The term “antigenic peptide or protein” will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a peptide, protein (or polyprotein) derived from a (antigenic) protein/polyprotein which may stimulate the body's adaptive immune system to provide an adaptive immune response. Therefore an “antigenic peptide or protein” comprises at least one epitope (as defined herein) or antigen (as defined herein) of the protein it is derived from (e.g., in the context of the invention, RSV peptide or protein, preferably RSV F protein or variants thereof).

Artificial nucleic acid: The terms “artificial nucleic acid” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to an artificial nucleic acid that does not occur naturally. An artificial nucleic acid may be a DNA molecule, an RNA molecule or a hybrid-molecule comprising DNA and RNA portions. Typically, artificial nucleic acids may be designed and/or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). In this context an artificial sequence is usually a sequence that may not occur naturally, i.e. it differs from the wild type sequence by at least one nucleotide. The term “wild type” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a sequence occurring in nature. Further, the term “artificial nucleic acid” is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of essentially identical molecules.

Artificial RNA: The term “artificial RNA” as used herein is intended to refer to an RNA that does not occur naturally. In other words, an artificial RNA may be understood as a non-natural nucleic acid molecule. Such RNA molecules may be non-natural due to its individual sequence (which does not occur naturally, e.g. G/C content modified coding sequence, UTRs) and/or due to other modifications, e.g. structural modifications of nucleotides which do not occur naturally. Typically, artificial RNA may be designed and/or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). In this context an artificial RNA sequence is usually a sequence that may not occur naturally, i.e. it differs from the wild type sequence by at least one nucleotide. The term “artificial RNA” is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of essentially identical molecules. Accordingly, it may relate to a plurality of essentially identical RNA molecules contained in an aliquot or a sample. In the context of the invention, the RNA of the invention is an artificial RNA as defined herein.

Cationic: Unless a different meaning is clear from the specific context, the term “cationic” means that the respective structure bears a positive charge, either permanently or not permanently but in response to certain conditions such as pH. Thus, the term “cationic” covers both “permanently cationic” and “cationisable”.

Cationisable: The term “cationisable” as used herein means that a compound, or group or atom, is positively charged at a lower pH and uncharged at a higher pH of its environment. Also in non-aqueous environments where no pH value can be determined, a cationisable compound, group or atom is positively charged at a high hydrogen ion concentration and uncharged at a low concentration or activity of hydrogen ions. It depends on the individual properties of the cationisable or polycationisable compound, in particular the pKa of the respective cationisable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged. In diluted aqueous environments, the fraction of cationisable compounds, groups or atoms bearing a positive charge may be estimated using the so-called Henderson-Hasselbalch equation which is well-known to a person skilled in the art. For example, in some embodiments, if a compound or moiety is cationisable, it is preferred that it is positively charged at a pH value of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably of a pH value of or below 9, of or below 8, of or below 7, most preferably at physiological pH values, e.g. about 7.3 to 7.4, i.e. under physiological conditions, particularly under physiological salt conditions of the cell in vivo. In other embodiments, it is preferred that the cationisable compound or moiety is predominantly neutral at physiological pH values, e.g. about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, the preferred range of pKa for the cationisable compound or moiety is about 5 to about 7.

Coding sequence/coding region: The terms “coding sequence” or “coding region” and the corresponding abbreviation “cds” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a sequence of several nucleotide triplets, which may be translated into a peptide or protein. A coding sequence in the context of the present invention is preferably an RNA sequence, consisting of a number of nucleotides that may be divided by three, which starts with a start codon and which preferably terminates with a stop codon.

Composition: In the context of the invention, a “composition” refers to any type of composition in which the specified ingredients (e.g. artificial RNA of the invention in association with LNP), may be incorporated, optionally along with any further constituents, usually with at least one pharmaceutically acceptable carrier or excipient. Thus, the composition may be a dry composition such as a powder or granules, or a solid unit such as a lyophilized form or a tablet. Alternatively, the composition may be in liquid form, and each constituent may be independently incorporated in dissolved or dispersed (e.g. suspended or emulsified) form.

Compound: As used herein, a “compound” means a chemical substance, which is a material consisting of molecules having essentially the same chemical structure and properties. For a small molecular compound, the molecules are typically identical with respect to their atomic composition and structural configuration. For a macromolecular or polymeric compound, the molecules of a compound are highly similar but not all of them are necessarily identical. For example, a segment of a polymer that is designated to consist of 50 monomeric units may also contain individual molecules with e.g. 48 or 53 monomeric units.

Derived from: The term “derived from” as used throughout the present specification in the context of a nucleic acid, i.e. for a nucleic acid “derived from” (another) nucleic acid, means that the nucleic acid, which is derived from (another) nucleic acid, shares at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, 81%, 82%, 83%, 84%, more preferably at least 85%, 86%, 87%, 88%, 89% even more preferably at least 90%, 91%, 92%, 93%, 94%, even more preferably at least 95%, 96%, 97%, and particularly preferably at least 98%, 99% sequence identity with the nucleic acid from which it is derived. The skilled person is aware that sequence identity is typically calculated for the same types of nucleic acids, i.e. for DNA sequences or for RNA sequences. Thus, it is understood, if a DNA is “derived from” an RNA or if an RNA is “derived from” a DNA, in a first step the RNA sequence is converted into the corresponding DNA sequence (in particular by replacing the uracils (U) by thymidines (T) throughout the sequence) or, vice versa, the DNA sequence is converted into the corresponding RNA sequence (in particular by replacing the thymidines (T) by uracils (U) throughout the sequence). Thereafter, the sequence identity of the DNA sequences or the sequence identity of the RNA sequences is determined.

Preferably, a nucleic acid “derived from” a nucleic acid also refers to nucleic acid, which is modified in comparison to the nucleic acid from which it is derived, e.g. in order to increase RNA stability even further and/or to prolong and/or increase protein production. It goes without saying that such modifications are preferred, which do not impair RNA stability, e.g. in comparison to the nucleic acid from which it is derived. In the context of amino acid sequences (e.g. antigenic peptides or proteins) the term “derived from” means that the amino acid sequence, which is derived from (another) amino acid sequence (e.g. RSV F protein), shares at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, 81%, 82%, 83%, 84%, more preferably at least 85%, 86%, 87%, 88%, 89% even more preferably at least 90%, 91%, 92%, 93%, 94%, even more preferably at least 95%, 96%, 97%, and particularly preferably at least 98%, 99% sequence identity with the amino acid sequence from which it is derived.

Thus, it is understood, if a antigenic peptides or protein is “derived from” an RSV fusion (F) protein, the antigenic peptides or protein that is “derived from” said RSV F protein may represent a variant or fragment of the RSV F protein, e.g. F0 (full-length precursor), F-del, F0_DSCav1, F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1, F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3 (as specified herein). Moreover, the antigenic peptides or protein that is “derived from” said RSV F proteins (e.g., F0, F-del, F0_DSCav1, F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1, F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3) may differ in the amino acid sequence, sharing a certain percentage of identity as defined above. Suitable further examples of RSV F proteins from which an antigenic peptides or protein may be “derived from” are provided in Table 1.

Epitope: The term “epitope” (also called “antigen determinant” in the art) as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to T cell epitopes and B cell epitopes. T cell epitopes or parts of the antigenic peptides or proteins may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 to about 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form. B cell epitopes are typically fragments located on the outer surface of (native) protein or peptide antigens, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form. Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides. In this context antigenic determinants can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain. In the context of the present invention, an epitope may be the product of translation of a provided artificial RNA as specified herein.

Fragment: The term “fragment” as used throughout the present specification in the context of a nucleic acid sequence or an amino acid sequence may typically be a shorter portion of a full-length sequence of e.g. a nucleic acid sequence or an amino acid sequence. Accordingly, a fragment, typically, consists of a sequence that is identical to the corresponding stretch within the full-length sequence. A preferred fragment of a sequence in the context of the present invention, consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 5%, 10%, 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the total (i.e. full-length) molecule from which the fragment is derived (e.g. RSV F protein). The term “fragment” as used throughout the present specification in the context of proteins or peptides may, typically, comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence (or its encoded nucleic acid molecule), N-terminally and/or C-terminally truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid molecule). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level. A sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide. In the context of antigens such fragment may have a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12 amino acids), or fragments as processed and presented by MHC class I molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form. Fragments of proteins or peptides (e.g. in the context of antigens) may comprise at least one epitope of those proteins or peptides. Furthermore also domains of a protein, like the extracellular domain, the intracellular domain or the transmembrane domain and shortened or truncated versions of a protein may be understood to comprise a fragment of a protein.

Heterologous: The terms “heterologous” or “heterologous sequence” as used throughout the present specification in the context of a nucleic acid sequence or an amino acid sequence refers to a sequence (e.g. DNA, RNA, amino acid) will be recognized and understood by the person of ordinary skill in the art, and is intended to refer to a sequence that is derived from another gene, from another allele, from another species. Two sequences are typically understood to be “heterologous” if they are not derivable from the same gene or in the same allele. I.e., although heterologous sequences may be derivable from the same organism, they naturally (in nature) do not occur in the same nucleic acid molecule, such as e.g. in the same RNA, or the same protein.

Humoral immune response: The terms “humoral immunity” or “humoral immune response” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to B-cell mediated antibody production and optionally to accessory processes accompanying antibody production. A humoral immune response may be typically characterized, e.g. by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

Identity (of a sequence): The term “identity” as used throughout the present specification in the context of a nucleic acid sequence or an amino acid sequence will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to the percentage to which two sequences are identical. To determine the percentage to which two sequences are identical, e.g. nucleic acid sequences or amino acid sequences as defined herein, preferably the amino acid sequences encoded by the artificial nucleic acid sequence as defined herein or the amino acid sequences themselves, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. a position of a first sequence may be compared with the corresponding position of the second sequence. If a position in the first sequence is occupied by the same component (residue) as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position. If insertions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the first sequence to allow a further alignment. If deletions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the second sequence to allow a further alignment. The percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence. The percentage to which two sequences are identical can be determined using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.

Immunogen, immunogenic: The terms “immunogen” or “immunogenic” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a compound that is able to stimulate/induce an immune response. Preferably, an immunogen is a peptide, polypeptide, or protein. An immunogen in the sense of the present invention is the product of translation of a provided artificial nucleic acid, preferably RNA, comprising at least one coding sequence encoding at least one antigenic peptide, protein derived from RSV as defined herein. Typically, an immunogen elicits an adaptive immune response.

Immune response: The term “immune response” will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response), or a combination thereof.

Immune system: The term “immune system” will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a system of the organism that may protect the organisms from infection. If a pathogen succeeds in passing a physical barrier of an organism and enters this organism, the innate immune system provides an immediate, but non-specific response. If pathogens evade this innate response, vertebrates possess a second layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered. According to this, the immune system comprises the innate and the adaptive immune system. Each of these two parts typically contains so called humoral and cellular components.

Innate immune system: The term “innate immune system” (also known as non-specific or unspecific immune system) will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a system typically comprising the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system may recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. The innate immune system may be, e.g. activated by ligands of Toll-like receptors (TLRs) or other auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1 to IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of human Toll-like receptor (e.g., TLR1 to TLR10), a ligand of murine Toll-like receptor, (e.g., TLR1 to TLR13), a ligand of a NOD-like receptor, a ligand of a RIG-I like receptor, an immunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), a CpG-DNA, an antibacterial agent, or an anti-viral agent.

Lipidoid compound: A lipidoid compound, also simply referred to as lipidoid, is a lipid-like compound, i.e. an amphiphilic compound with lipid-like physical properties. In the context of the present invention the term lipid is considered to encompass lipidoid compounds.

Monovalent vaccine, monovalent composition: The terms “monovalent vaccine”, “monovalent composition” “univalent vaccine” or “univalent composition” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a composition or a vaccine comprising only one antigen from a virus. Accordingly, said vaccine or composition comprises only one RNA species encoding a single antigen for a single organism. The term “monovalent vaccine” includes the immunization against a single valence. In the context of the invention, a monovalent RSV vaccine or composition would comprise an artificial RNA encoding one single antigenic peptide or protein derived from one specific RSV (e.g. RSV F).

Nucleic acid: The terms “nucleic acid” or “nucleic acid molecule” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a molecule comprising, preferably consisting of nucleic acid components. The term nucleic acid molecule preferably refers to DNA or RNA molecules. It is preferably used synonymous with the term polynucleotide. Preferably, a nucleic acid or a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone. The term “nucleic acid molecule” also encompasses modified nucleic acid molecules, such as base-modified, sugar-modified or backbone-modified DNA or RNA molecules as defined herein.

Nucleic acid sequence/RNA sequence/amino acid sequence: The terms “nucleic acid sequence”, “RNA sequence” or “amino acid sequence” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to particular and individual order of the succession of its nucleotides or amino acids respectively.

Permanently cationic: The term “permanently cationic” as used herein will be recognized and understood by the person of ordinary skill in the art, and means, for example, that the respective compound, or group or atom, is positively charged at any pH value or hydrogen ion activity of its environment. Typically, the positive charge is results from the presence of a quaternary nitrogen atom. Where a compound carries a plurality of such positive charges, it may be referred to as permanently polycationic, which is a subcategory of permanently cationic.

Pharmaceutically effective amount: The terms “pharmaceutically effective amount” or “effective amount” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to an amount of a compound (e.g. the artificial RNA of the invention) that is sufficient to induce a pharmaceutical effect, such as, in the context of the invention, an immune response (e.g. against an antigenic peptide, protein, polyprotein as defined herein).

Polyvalent/multivalent vaccine, polyvalent/multivalent composition: The terms “polyvalent vaccine”, “polyvalent composition” “multivalent vaccine” or “multivalent composition” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a composition or a vaccine comprising antigens from more than one strain of a virus, or comprising different antigens of the same virus, or any combination thereof. The terms describe that said vaccine or composition has more than one valence. In the context of the invention, a polyvalent RSV vaccine would comprise a vaccine comprising an artificial RNA encoding antigenic peptides or proteins derived from several different RSV strains or comprising artificial RNA encoding different antigens from the same RSV strain, or a combination thereof. In preferred embodiment, a polyvalent RSV vaccine or composition comprises more than one, preferably 2, 3, 4 or even more different artificial RNA species each encoding at least one different antigenic peptide or protein of RSV (e.g. RSV F and RSV M or RSV F and RSV N). Methods to produce polyvalent mRNA vaccines are disclosed in the PCT application PCT/EP2016/082487 or in published patent application WO2017/1090134A1.

Stabilized nucleic acid molecule” or “stabilized RNA: The term “stabilized nucleic acid molecule” or “stabilized RNA” refer to a nucleic acid molecule, preferably an RNA molecule that is modified such, that it is more stable to disintegration or degradation, e.g., by environmental factors or enzymatic digest, such as by an exo- or endonuclease degradation, than the nucleic acid molecule without the modification. Preferably, a stabilized nucleic acid molecule, e.g. stabilized RNA, in the context of the present invention is stabilized in a cell, such as a prokaryotic or eukaryotic cell, preferably in a mammalian cell, such as a human cell. The stabilization effect may also be exerted outside of cells, e.g. in a buffer solution etc., for example, in a manufacturing process for a pharmaceutical composition comprising the stabilized nucleic acid molecule.

T-cell responses: The terms “cellular immunity” or “cellular immune response” or “cellular T-cell responses” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. In more general terms, cellular immunity is not based on antibodies, but on the activation of cells of the immune system. Typically, a cellular immune response may be characterized e.g. by activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in cells, e.g. specific immune cells like dendritic cells or other cells, displaying epitopes of foreign antigens on their surface. In the context of the invention, the antigen is provided by the artificial RNA encoding at least one antigenic peptide or protein derived from RSV, suitably inducing T-cell responses. The artificial RNA, the composition, the vaccine of the invention advantageously elicit cellular T-cell responses against RSV F antigens.

Variant (of a sequence): The term “variant” as used throughout the present specification in the context of a nucleic acid sequence will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a variant of nucleic acid sequences which forms the basis of a nucleic acid sequence. For example, a variant nucleic acid sequence may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the nucleic acid sequence from which the variant is derived. Preferably, a variant of a nucleic acid sequence is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the nucleic acid sequence the variant is derived from. Preferably, the variant is a functional variant. A “variant” of a nucleic acid sequence may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity over a stretch of 10, 20, 30, 50, 75 or 100 nucleotide of such nucleic acid sequence.

The term “variant” as used throughout the present specification in the context of proteins or peptides will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a proteins or peptide variant having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s). Preferably, these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific antigenic property. “Variants” of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e. non-mutated physiological, sequence. Those amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined herein. Substitutions in which amino acids, which originate from the same class, are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function. This means that e.g. an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra). A “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide. Preferably, a variant of a protein comprises a functional variant of the protein, which means that the variant exerts the same effect or functionality as the protein it is derived from.

3′-untranslated region, 3′-UTR element, 3′-UTR: The term “3′-untranslated region” or “3′-UTR element” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a part of a nucleic acid molecule, which is located 3′ (i.e. “downstream”) of a coding sequence and which is typically not translated into protein. Usually, a 3′-UTR is the part of an mRNA which is located between the coding sequence (cds) and the poly(A) sequence of the mRNA. In the context of the invention, the term 3′-UTR may also comprise elements, which are not encoded in the DNA template, from which an artificial RNA is transcribed, but which are added after transcription during maturation, e.g. a poly(A) sequence.

5-untranslated region, 5′-UTR element, 5′-UTR: The term “5′-untranslated region (5′-UTR)” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a part of a nucleic acid molecule, which is located 5′ (i.e. “upstream”) of a coding sequence and which is not translated into protein. A 5′-UTR is typically understood to be a particular section of messenger RNA (mRNA), which is located 5′ of the coding sequence of the mRNA. Typically, the 5′-UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the coding sequence. Preferably, the 5′-UTRs have a length of more than 20, 30, 40 or 50 nucleotides. The 5′-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites. The 5′-UTR may be post-transcriptionally modified, for example by addition of a 5′-cap.

5-terminal oligopyrimidine tract (TOP), TOP-UTR: The term “5′-terminal oligopyrimidine tract (TOP)” has to be understood as a stretch of pyrimidine nucleotides located in the 5-terminal region of a nucleic acid molecule, such as the 5-terminal region of certain mRNA molecules or the 5-terminal region of a functional entity, e.g. the transcribed region, of certain genes. The sequence starts with a cytidine, which usually corresponds to the transcriptional start site, and is followed by a stretch of usually about 3 to 30 pyrimidine nucleotides. For example, the TOP may comprise 3-30 or even more nucleotides. The pyrimidine stretch and thus the 5′-TOP ends one nucleotide 5′ to the first purine nucleotide located downstream of the TOP. Messenger RNA that contains a 5′-terminal oligopyrimidine tract is often referred to as TOP mRNA. Accordingly, genes that provide such messenger RNAs are referred to as TOP genes. The term “TOP motif” or “5′-TOP motif” has to be understood as a nucleic acid sequence which corresponds to a 5′-TOP as defined above. Thus, a TOP motif in the context of the present invention is preferably a stretch of pyrimidine nucleotides having a length of 3-30 nucleotides. Preferably, the TOP-motif consists of at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 nucleotides, most preferably at least 8 pyrimidine nucleotides, wherein the stretch of pyrimidine nucleotides preferably starts at its 5′-end with a cytosine nucleotide. In TOP genes and TOP mRNAs, the TOP-motif preferably starts at its 5′-end with the transcriptional start site and ends one nucleotide 5′ to the first purine residue in said gene or mRNA. A TOP motif in the sense of the present invention is preferably located at the 5′-end of a sequence which represents a 5′-UTR or at the 5′-end of a sequence which codes for a 5′-UTR. Thus, preferably, a stretch of 3 or more pyrimidine nucleotides is called “TOP motif” in the sense of the present invention if this stretch is located at the 5′-end of a respective sequence, such as the artificial nucleic acid, the 5′-UTR element of the artificial nucleic acid, or the nucleic acid sequence which is derived from the 5′-UTR of a TOP gene as described herein. In other words, a stretch of 3 or more pyrimidine nucleotides, which is not located at the 5′-end of a 5′-UTR or a 5′-UTR element but anywhere within a 5′-UTR or a 5′-UTR element, is preferably not referred to as “TOP motif”. In some embodiments, the nucleic acid sequence of the 5′-UTR element, which is derived from a 5′-UTR of a TOP gene, terminates at its 3′-end with a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of the start codon (e.g. A(U/T)G) of the gene or RNA it is derived from. Thus, the 5′-UTR element does not comprise any part of the protein coding sequence. Thus, preferably, the only protein coding part of the at least one nucleic acid sequence, particularly of the RNA sequence, is provided by the coding sequence.

SHORT DESCRIPTION OF THE INVENTION

The present invention is based on the inventor's surprising finding that at least one peptide or protein derived from of a Respiratory syncytial virus (RSV) F protein encoded by the artificial RNA of the invention can efficiently be expressed in a mammalian cell. Even more unexpected, the inventors showed that the artificial RNA of the invention can induce specific functional and protective immune responses in e.g. cotton rats (see e.g. Example 2, 3). Through different optimizations in RSV F antigen design, the immune responses could be further improved. In addition, the expression of the RSV F antigen encoded by the artificial nucleic RNA could be increased by selecting suitable heterologous 5′ untranslated regions (UTRs) and suitable heterologous 3 untranslated regions (UTRs) (see e.g. Example 4). Advantageously, said artificial RNA of the invention comprising advantageous 3-UTR/5′-UTR combinations induce very efficient antigen-specific immune responses against the encoded RSV F. Further, artificial RNA of the invention comprised in lipid nanoparticles (LNPs) very efficiently induces antigen-specific immune responses against RSV F at a very low dosages and dosing regimen (see e.g. Example 3). Further, e.g. Example 8 and Example 12 provide compositions/vaccines comprising a further artificial RNA encoding a further antigen wherein said artificial RNA encoding a further antigen suitably elicits or enhances T-cell responses and results in a Th1-biased immune response, which is considered to be an important prerequisite for a potential RSV vaccine (Th2-biased responses have been associated with enhanced respiratory disease (ERD) in animal models). Furthermore the compositions are suitable to induce T-cell responses. Accordingly, the artificial RNA, and the composition/vaccine comprising said artificial RNA of the invention are suitable for eliciting an immune response against RSV F in a mammalian subject. The artificial RNA and the composition/vaccine comprising said artificial RNA is therefore suitable for use as a vaccine, e.g. as a human vaccine, e.g. as a vaccine for pregnant women or infants.

In a first aspect, the present invention provides an artificial nucleic acid, preferably an artificial RNA comprising at least one 5′ untranslated region (UTR) and/or at least one 3′ untranslated region (UTR), and at least one coding sequence operably linked to said 3′-UTR and/or 5′-UTR encoding at least one antigenic peptide or protein derived from RSV F protein or a fragment or variant thereof.

In preferred embodiments, the artificial RNA comprises at least one nucleic acid sequence derived from a 3′-UTR of a gene selected from an ALB7 gene, an alpha-globin gene, a PSMB3, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or a variant of any one of these genes

In preferred embodiments, the artificial RNA comprises at least one nucleic acid sequence derived from a 5′-UTR of gene selected from a RPL32 gene, a HSD17B4, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4 and UBQLN2, or from a homolog, a fragment or variant of any one of these genes.

Suitably, the artificial RNA of the invention comprises at least one coding sequence encoding at least one antigenic peptide or protein derived from a RSV F protein operably linked to a 3′-UTR and a 5′-UTR selected from a-1 (HSD17B4/PSMB3), a-2 (Ndufa4/PSMB3), a-3 (SLC7A3/PSMB3), a-4 (NOSIP/PSMB3), a-5 (MP68/PSMB3), b-1 (UBQLN2/RPS9), b-2 (ASAH1/RPS9), b-3 (HSD17B4/RPS9), b-4 (HSD17B4/CASP1), b-5 (NOSIP/COX6B1), c-1 (NDUFA4/RPS9), c-2 (NOSIP/NDUFA1), c-3 (NDUFA4/COX6B1), c-4 (NDUFA4/NDUFA1), c-5 (ATP5A1/PSMB3), d-1 (Rpl31/PSMB3), d-2 (ATP5A1/CASP1), d-3 (SLC7A3/GNAS), d-4 (HSD17B4/NDUFA1), d-5 (Slc7a3/Ndufa1), e-1 (TUBB4B/RPS9), e-2 (RPL31/RPS9), e-3 (MP68/RPS9), e-4 (NOSIP/RPS9), e-5 (ATP5A1/RPS9), e-6 (ATP5A1/COX6B1), f-1 (ATP5A1/GNAS), f-2 (ATP5A1/NDUFA1), f-3 (HSD17B4/COX6B1), f-4 (HSD17B4/GNAS), f-5 (MP68/COX6B1), g-1 (MP68/NDUFA1), g-2 (NDUFA4/CASP1), g-3 (NDUFA4/GNAS), g-4 (NOSIP/CASP1), g-5 (RPL31/CASP1), h-1 (RPL31/COX6B1), h-2 (RPL31/GNAS), h-3 (RPL31/NDUFA1), h-4 (Slc7a3/CASP1), h-5 (SLC7A3/COX6B1), i-1 (SLC7A3/RPS9), i-2 (RPL32/ALB7), or i-3 (a-globin gene), wherein a-1 (HSD17B4/PSMB3), a-4 (NDUFA4/PSMB3), c-1 (NDUFA4/RPS9), e-4 NOSIP/RPS9), g-2 (NDUFA4/CASP1), i-2 (RPL32/ALB7), or i-3 (alpha-globin) are particularly preferred.

The at least one antigenic peptide or protein derived from RSV F protein may be a full-length F protein (referred to as “F0”, aa 1-574) or an F protein with deleted C-terminus (referred to as “F-del”, aa 1-553), or a fragment or a variant thereof.

The at least one antigenic peptide or protein may additionally comprise a mutation that stabilizes the antigen in pre-conformation state/pre-fusion conformation, preferably a DSCav1 mutation (S155C, S290C, S190F, and V207L) or a fragment or a variant, or a functional variant thereof (referred to as “DSCav1”, e.g. “F0_DSCav1” or “F-del_DSCav1”).

The at least one antigenic peptide or protein may be a fusion protein comprising the two subunits, F1 and F2 of mature F into a single chain, connected via a linker (GS) to enhance stability of the protein (F(1-103)-GS-F(145-574); F(1-103)-GS-F(145-553)).

The protein comprising the two subunits of mature F into a single chain (referred to as “F2-linker-F1”), e.g. (F(1-103)-GS-F(145-574); F(1-103)-GS-F(145-553) may additionally comprise a DSCav1 mutation (herein referred to as “mut0”).

The protein comprising the two subunits of mature F into a single chain (referred to as “F2-linker-F1”) may additionally to the DSCav1 mutation comprise at least one further mutation that promotes inter-protomer disulphide bonds, wherein the mutations may be selected from (S46G, A149C, S215P, Y458C, K465Q; herein referred to as “mut1”), (S46G, E92D, A149C, S215P, Y458C, K465Q; herein referred to as “mut2”), or (S46G, N671, E92D, A149C, S215P, Y458C, K465Q; herein referred to as “mut3”), (A149C, Y458C; herein referred to as “mut4”), (N183GC, N428C; herein referred to as “mut5”), (Q98C, Q361C, S46G, E92D, L95M, S215P, I217P, I221M, R429K, K465Q; herein referred to as “mut6”), (Q98C, Q361C, L95M, I221M, R429K; herein referred to as “mut7”), or (N183GC, N428C, S46G, N671, E92D, S215P, K465Q; herein referred to as “mut8”) or a fragment or a variant, or a functional variant thereof.

The at least one antigenic peptide or protein derived from RSV F protein encoded by the artificial RNA of the invention may be selected from F0, F-del, F0_DSCav1, F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3, F_DSCav1_mut4, F_DSCav1_mut5, F_DSCav1_mut6, F_DSCav1_mut7, F_DSCav1_mut8, F_DSCav1_mut0, F-del_DSCav1, F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut4, F-del_DSCav1_mut5, F-del_DSCav1_mut6, F-del_DSCav1_mut7, F-del_DSCav1_mut8, F-del_DSCav1_mut0 are preferred.

The at least one coding sequence may encode at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909, 8279-9683, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106, 19475 or a fragment or variant of any of these sequences.

Preferably, the artificial RNA may comprise a coding sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 69-77, 484-492, 899-906, 1268-1275, 1637-1644, 2006-2013, 2375-2382, 2744-2751, 3113-3120, 3482-3489, 3851-3858, 4220-4227, 4589-4596, 4958-4965, 5327-5334, 5696-5703, 6065-6072, 6434-6441, 6803-6810, 7172-7179, 7541-7548, 7910-7917, 21363-21384, 11727-11734, 12096-12103, 12465-12472, 12834-12841, 13941-13948, 14310-14317, 14679-14686, 15048-15055, 15417-15424, 15786-15793, 13203-13210, 13572-13579, 16155-16162, 16524-16531, 16893-16900, 17262-17269, 17631-17638, 18000-18007, 18369-18376, 18738-18745, 19107-19114, 19476-19483. 21389-21410 or a fragment or variant of any of these sequences.

The artificial RNA may comprise a codon modified coding sequence selected from C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified coding sequence, and G/C optimized coding sequence, or any combination thereof.

The artificial RNA may be an mRNA, a viral RNA, self-replicating RNA, a circular RNA, or a replicon RNA. In preferred embodiments, the artificial RNA is an mRNA.

The artificial RNA, preferably mRNA, may further comprise at least one selected from a cap structure, a poly(A)sequence, a poly(C)sequence, a histone-stem loop, and/or a 3′-terminal sequence element.

The artificial RNA of the invention preferably comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 78-482, 11735-12094, 21415-21417, 21561-21563, 21489, 21490, 21635, 21636 or a fragment or variant of any of these (encoding F0), SEQ ID NOs: 493-897, 12104-12463, 21418-21420, 21564-21566, 21491, 21492, 21637, 21638 or a fragment or variant of any of these (encoding F-del), SEQ ID NOs: 907-1266, 12473-12832, 21421-21423, 21567-21569, 21493-21495, 21639-21641 or a fragment or variant of any of these (encoding F0_DSCav1), SEQ ID NOs: 1276-1635, 8278, 12842-13201, 21424-21426, 21570-21572, 21496-21498, 21642-21644 or a fragment or variant of any of these (encoding F-del_DSCav1), SEQ ID NOs: 1645-2004, 13949-14308, 21433-21435, 21579-21581, 21505-21507, 21651-21653 or a fragment or variant of any of these (encoding F_DSCav1_mut1), SEQ ID NOs: 2014-2373, 14318-14677, 21436-21438, 21582-21584, 21508-21510, 21654-21656 or a fragment or variant of any of these (encoding F-del_DSCav1_mut1), SEQ ID NOs: 2383-2742, 14687-15046, 21439-21441, 21585-21587, 21511-21513, 21657-21659 or a fragment or variant of any of these (encoding F_DSCav1_mut2), SEQ ID NOs: 2752-3111, 15056-15415, 21442-21444, 21588-21590, 21514-21516, 21660-21662 or a fragment or variant of any of these (encoding F-del_DSCav1_mut2), SEQ ID NOs: 3121-3480, 15425-15784, 21445-21447, 21591-21593, 21517-21519, 21663-21665 or a fragment or variant of any of these (encoding F_DSCav1_mut3), SEQ ID NOs: 3490-3849, 15794-16153, 21448-21450, 21594-21596, 21520-21522, 21666-21668 or a fragment or variant of any of these (encoding F-del_DSCav1_mut3), SEQ ID NOs: 3859-4218, 13211-13570, 21427-21429, 21573-21575, 21499-21501, 21645-21647 or a fragment or variant of any of these (encoding F_DSCav1_mut0), SEQ ID NOs: 4228-4587, 13580-13939, 21430-21432, 21576-21578, 21502-21504, 21648-21650 or a fragment or variant of any of these (encoding F-del_DSCav1_mut0), SEQ ID NOs: 4597-4956, 16163-16522, 21451-21453, 21597-21599, 21523-21525, 21669-21671 or a fragment or variant of any of these (encoding F_DSCav1_mut4), SEQ ID NOs: 4966-5325, 16532-16891, 21454-21456, 21600-21602, 21526-21528, 21672-21674 or a fragment or variant of any of these (encoding F-del_DSCav1_mut4), SEQ ID NOs: 5335-5694, 16901-17260, 21457-21459, 21603-21605, 21529-21531, 21675-21677 or a fragment or variant of any of these (encoding F_DSCav1_mut5), SEQ ID NOs: 5704-6063, 17270-17629, 21460-21462, 21606-21608, 21532-21534, 21678-21680 or a fragment or variant of any of these (encoding F-del_DSCav1_mut5), SEQ ID NOs: 6073-6432, 17639-17998, 21463-21465, 21609-21611, 21535-21537, 21681-21683 or a fragment or variant of any of these (encoding F_DSCav1_mut6), SEQ ID NOs: 6442-6801, 18008-18367, 21466-21468, 21612-21614, 21538-21540, 21684-21686 or a fragment or variant of any of these (encoding F-del_DSCav1_mut6), SEQ ID NOs: 6811-7170, 18377-18736, 21469-21471, 21615-21617, 21541-21543, 21687-21689 or a fragment or variant of any of these (encoding F_DSCav1_mut7), SEQ ID NOs: 7180-7539, 18746-19105, 21472-21474, 21618-21620, 21544-21546, 21690-21692 or a fragment or variant of any of these (encoding F-del_DSCav1_mut7), SEQ ID NOs: 7549-7908, 19115-19474, 21475-21477, 21621-21623, 21547-21549, 21693-21695 or a fragment or variant of any of these (encoding F_DSCav1_mut8), or SEQ ID NOs: 7918-8277, 19484-19843, 21478-21480, 21624-21626, 21550-21552, 21696-21698 or a fragment or variant of any of these (encoding F-del_DSCav1_mut8).

In a second aspect, the present invention provides a composition comprising the artificial RNA of the first aspect.

In preferred embodiments, the composition comprising the artificial RNA of the first aspect comprises at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV selected from matrix protein M, nucleoprotein N, M2-1 protein, and/or phosphoprotein P or combinations thereof.

Matrix protein M, nucleoprotein N, M2-1 protein, M2-2 protein, and/or phosphoprotein P are suitable T-cell antigens and may promote efficient T-cell responses of the composition or vaccine when administered to a subject.

The further artificial RNA may comprise a coding sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 9685-9692, 10135-10142, 10638-10645, 11184-11191, 21385-21388, 19845-19852, 20214-20221, 20583-20590, 20952-20959, 21411-21414 or a fragment or variant of any of these sequences.

Suitably, said further artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 9693-10052, 10143-10502, 10646-11005, 11192-11551, 19853-20212, 20222-20581, 20591-20950, 20960-21319, 21481-21488, 21627-21634, 21553-21560, 21699-21706 or a fragment or variant of any of these sequences.

Suitably, the composition may comprise the artificial RNA of the invention complexed with, encapsulated in, or associated with one or more lipids, thereby forming lipid nanoparticles.

The composition may preferably comprise the artificial RNA of the invention complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein the LNP essentially consists of

(i) at least one cationic lipid as defined herein, preferably a lipid of formula (III), more preferably lipid III-3;

(ii) a neutral lipid as defined herein, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);

(iii) a steroid or steroid analogue as defined herein, preferably cholesterol; and

(iv) a PEG-lipid as defined herein, e.g. PEG-DMG or PEG-cDMA, preferably a PEGylated lipid of formula (IVa); wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.

The present invention also concerns a RSV vaccine comprising said artificial RNA or said composition.

The present invention is also directed to the use of the artificial RNA, the composition and the vaccine in treatment or prophylaxis of an infection with RSV.

In particular, the present invention is directed to the use of the artificial RNA, the composition and the vaccine in treatment or prophylaxis of an infection with RSV or a disorder related to such an infection.

The invention further concerns a method of treating or preventing a disorder or a disease in a subject, first and second medical uses of the artificial RNA, compositions and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the artificial RNA, compositions and vaccines.

DETAILED DESCRIPTION OF THE INVENTION

The present application is filed together with a sequence listing in electronic format, which is part of the description of the present application (WIPO standard ST.25). The information contained in the electronic format of the sequence listing filed together with this application is incorporated herein by reference in its entirety. Where reference is made herein to a “SEQ ID NO” the corresponding nucleic acid sequence or amino acid (aa) sequence in the sequence listing having the respective identifier is referred to. For many sequences, the sequence listing also provides additional detailed information, e.g. regarding certain structural features, sequence optimizations, GenBank identifiers, or additional detailed information regarding its coding capacity. In particular, such information is provided under numeric identifier <223> in the WIPO standard ST.25 sequence listing. Accordingly, information provided under said numeric identifier <223> is explicitly included herein in its entirety and has to be understood as integral part of the description of the underlying invention.

Artificial Nucleic Acid:

In a first aspect, the invention relates to an artificial nucleic acid comprising

-   a) at least one heterologous 5′ untranslated region (5′-UTR) and/or     at least one heterologous 3′ untranslated region (3′-UTR); and -   b) at least one coding sequence operably linked to said 3′-UTR     and/or 5′-UTR encoding at least one antigenic peptide or protein     derived from a Respiratory syncytial virus (RSV) or a fragment or     variant thereof.

In a preferred embodiment of the first aspect, the invention relates to an artificial RNA, preferably an RNA suitable for vaccination, comprising

-   a) at least one heterologous 5′ untranslated region (5′-UTR) and/or     at least one heterologous 3′ untranslated region (3′-UTR); and -   b) at least one coding sequence operably linked to said 3′-UTR     and/or 5′-UTR encoding at least one antigenic peptide or protein     derived from a RSV fusion (F) protein or a fragment or variant     thereof.

In general, the RNA of the invention may be composed of a protein-coding region, and 5′- and/or 3-untranslated regions (UTRs). The 3′-UTR is variable in sequence and size; it spans between the stop codon and the poly(A) tail. Importantly, the 3′-UTR sequence harbors several regulatory motifs that determine RNA turnover, stability and localization, and thus governs many aspects of post-transcriptional regulation. In medical application of RNA (e.g. immunotherapy applications, vaccination) the regulation of RNA translation into protein is of paramount importance to therapeutic safety and efficacy. The present inventors surprisingly discovered that certain combinations of 3-UTRs and/or 5′-UTRs act in concert to synergistically enhance the expression of operably linked nucleic acid sequences encoding RSV antigenic peptides or proteins. Artificial RNA molecules harboring the inventive UTR combinations advantageously enable the rapid and transient expression of high amounts of RSV antigenic peptides or proteins derived from RSV F. Accordingly, the artificial RNA provided herein is particularly useful and suitable for various applications in vivo, including the vaccination against RSV.

Suitably, the artificial RNA may comprise at least one heterologous 5′-UTR and/or at least one heterologous 3′-UTR. In this context, an UTR of the invention comprises or consists of a nucleic acid sequence derived from a 5′-UTR or a 3′-UTR of any naturally occurring gene or a fragment, a homolog or a variant thereof. Preferably, a 5-UTR or a 3′-UTR of the invention is heterologous to the at least one coding sequence encoding the at least one antigenic peptide or protein derived from RSV F. Suitable heterologous 5′-UTRs or heterologous 3′-UTRs are derived from naturally occurring genes (that are not derived from RSV). In other embodiments, synthetically engineered 5′-UTRs or 3-UTRs may be used in the context of the present invention.

In preferred embodiments, the at least one artificial RNA comprises at least one heterologous 3′-UTR.

Preferably, the at least one heterologous 3′-UTR comprises or consists of a nucleic acid sequence derived from a 3′-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of a 3′-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene.

Preferably the artificial RNA of the present invention comprises a 3′-UTR, which may be derivable from a gene that relates to an RNA with an enhanced half-life (that provides a stable RNA), for example a 3′-UTR as defined and described below.

Preferably, the at least one heterologous 3′-UTR comprises a nucleic acid sequence derived from a 3′-UTR of a gene, which preferably encodes a stable mRNA, or from a homolog, a fragment or a variant of said gene.

In preferred embodiments of the first aspect, the artificial RNA of the invention comprises at least one heterologous 3′-UTR, wherein the at least one heterologous 3′-UTR comprises a nucleic acid sequence derived from a 3′-UTR of a gene selected from PSMB3, ALB7, alpha-globin (referred to as “muag”), CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or variant of any one of these genes.

ALB7-derived 3′-UTR: In preferred embodiments, the 3′-UTR comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a vertebrate albumin gene or from a variant thereof, preferably from the 3′-UTR of a mammalian albumin gene or from a variant thereof, more preferably from the 3′-UTR of a human albumin gene or from a variant thereof, even more preferably from the 3′-UTR of the human albumin gene according to GenBank Accession number NM_000477.5, or from a homolog, fragment or variant thereof. Accordingly, the artificial RNA of the invention may comprise a 3′-UTR derived from a ALB7 gene, wherein said 3′-UTR derived from a ALB7 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35 or 36 or a fragment or a variant thereof.

Alpha-globin gene-derived 3′-UTR: In preferred embodiments, the 3′-UTR comprises or consists of a nucleic acid sequence which is derived from the 3′-UTR of a vertebrate alpha-globin gene (referred to as “muag”) or from a variant thereof, preferably from the 3′-UTR of a mammalian alpha-globin or from a variant thereof, more preferably from the 3′-UTR of a human alpha-globin gene or from a variant thereof, even more preferably from the 3′-UTR of the human alpha-globin gene. Accordingly, the RNA of the invention may comprise a 3′-UTR derived from a alpha-globin gene, wherein said 3′-UTR derived from a alpha-globin gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37 or 38 or a fragment or a variant thereof.

PSMB3-derived 3′-UTR: The artificial RNA of the invention may comprise a 3′-UTR which is derived from a 3′-UTR of a gene encoding a proteasome subunit beta type-3 (PSMB3) protein, or a homolog, variant, fragment or derivative thereof. Such 3′-UTRs preferably comprise or consist of a nucleic acid sequences derived from the 3′-UTR of a proteasome subunit beta type-3 (PSMB3) gene, preferably from a vertebrate, more preferably a mammalian, most preferably a human proteasome subunit beta type-3 (PSMB3) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a proteasome subunit beta type-3 (PSMB3) protein corresponding to a human proteasome subunit beta type-3 (PSMB3) protein (UniProt Ref. No. P49720, entry version #183 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 3′-UTR derived from a PSMB3 gene, wherein said 3′-UTR derived from a PSMB3 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23 or 24 or a fragment or a variant thereof.

CASP1-derived 3′-UTR: The artificial RNA of the invention may comprise a 3′-UTR which is derived from a 3′-UTR of a gene encoding a Caspase-1 (CASP1) protein, or a homolog, variant, fragment or derivative thereof. Such 3′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 3′-UTR of a Caspase-1 (CASP1) gene, preferably from a vertebrate, more preferably a mammalian, most preferably a human Caspase-1 (CASP1) gene, or a homolog, variant, fragment or derivative thereof. Accordingly, the RNA of the invention may comprise a 3′-UTR derived from a CASP1 gene, wherein said 3′-UTR derived from a CASP1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25 or 26 or a fragment or a variant thereof.

COX6B1-derived 3′-UTR: The artificial RNA of the invention may comprise a 3′-UTR which is derived from a 3′-UTR of a COX6B1 gene encoding a cytochrome c oxidase subunit 6B1 (COX6B1) protein, or a homolog, variant, fragment or derivative thereof. Such 3′-UTRs preferably comprise or consist of a nucleic acid sequence which is derived from the 3′-UTR of a cytochrome c oxidase subunit 6B1 (COX6B1) gene, preferably from a vertebrate, more preferably a mammalian, most preferably a human cytochrome c oxidase subunit 6B1 (COX6B1) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a cytochrome c oxidase subunit 681 (COX6B1) protein corresponding to a human cytochrome c oxidase subunit 6B1 (COX6B1) protein (UniProt Ref. No. P14854, entry version #166 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 3′-UTR derived from a COX6B1 gene, wherein said 3′-UTR derived from a COX6B1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27 or 28 or a fragment or a variant thereof.

GNAS-derived 3′-UTR: The artificial RNA of the invention may comprise a 3′-UTR which is derived from a 3′-UTR of a GNAS gene encoding a Guanine nucleotide-binding protein G(s) subunit alpha isoforms short (GNAS) protein, or a homolog, variant, fragment or derivative thereof. Such 3-UTRs preferably comprise or consist of a nucleic acid sequence which is derived from the 3′-UTR of a Guanine nucleotide-binding protein G(s) subunit alpha isoforms short (GNAS) gene, preferably from a vertebrate, more preferably a mammalian Guanine nucleotide-binding protein G(s) subunit alpha isoforms short (GNAS) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a Guanine nucleotide-binding protein G(s) subunit alpha isoforms short (GNAS) protein corresponding to a human Guanine nucleotide-binding protein G(s) subunit alpha isoforms short (GNAS) protein (UniProt Ref. No. P63092, entry version #153 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 3′-UTR derived from a GNAS gene, wherein said 3′-UTR derived from a GNAS gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or 30 or a fragment or a variant thereof.

NDUFA1-derived 3′-UTR: The artificial RNA of the invention may comprise a 3′-UTR which is derived from a 3′-UTR of a gene encoding a NADH dehydrogenase [ubiquinone] 1 alpha sub complex subunit 1 (NDUFA1) protein, or a homolog, variant, fragment or derivative thereof. Such 3′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 3′-UTR of a NADH dehydrogenase [ubiquinone] 1 alpha sub complex subunit 1 (NDUFA1) gene, preferably from a vertebrate, more preferably a mammalian NADH dehydrogenase [ubiquinone]1 alpha sub complex subunit 1 (NDUFA1) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a NADH dehydrogenase [ubiquinone] 1 alpha sub complex subunit 1 (NDUFA1) protein corresponding to a human NADH dehydrogenase [ubiquinone] 1 alpha sub complex subunit 1 (NDUFA1) protein (UniProt Ref. No. 015239, entry version #152 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 3′-UTR derived from a NDUFA1 gene, wherein said 3′-UTR derived from a NDUFA1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31 or 32 or a fragment or a variant thereof.

RPS9-derived 3′-UTR: The artificial RNA of the invention may comprise a 3′-UTR which is derived from a 3′-UTR of a gene encoding a 40S ribosomal protein S9 (RPS9) protein, or a homolog, variant, fragment or derivative thereof. Such 3′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 3′-UTR of a 40S ribosomal protein S9 (RPS9) gene, preferably from a vertebrate, more preferably a mammalian, most preferably a human 40S ribosomal protein S9 (RPS9) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a 40S ribosomal protein S9 (RPS9) protein corresponding to a human 40S ribosomal protein S9 (RPS9) protein (UniProt Ref. No. P46781, entry version #179 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 3′-UTR derived from a RPS9 gene, wherein said 3′-UTR derived from a RPS9 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 or 34 or a fragment or a variant thereof.

Further 3′-UTRs: In embodiments, the artificial RNA as defined herein comprises a 3′-UTR as described in WO2016/107877. In this context, the disclosure of WO2016/107877 relating to 3′-UTR sequences is herewith incorporated by reference. Particularly suitable 3′-UTRs are SEQ ID NOs: 1 to 24 and SEQ ID NOs: 49 to 318 of patent application WO2016/107877, or fragments or variants of these sequences. Accordingly, the 3′-UTRs of the artificial RNA of the present invention may comprise or consists of a corresponding RNA sequence of the nucleic acid sequence according SEQ ID NOs: 1 to 24 and SEQ ID NOs: 49 to 318 of the patent application WO2016/107877. In other embodiments, the artificial RNA as defined herein comprises a 3′-UTR as described in WO2017/036580. In this context, the disclosure of WO2017/036580 relating to 3′-UTR sequences is herewith incorporated by reference. Particularly suitable 3-UTRs are SEQ ID NOs: 152 to 204 of the patent application WO2017/036580, or fragments or variants of these sequences. Accordingly, the 3′-UTR of the artificial RNA of the present invention may comprise or consist of a corresponding RNA sequence of the nucleic acid sequence according SEQ ID NOs: 152 to 204 of the patent application WO2017/036580.

According to preferred embodiments the artificial RNA comprises at least one heterologous 5′-UTR.

In preferred embodiments, the at least one artificial nucleic acid as defined herein, particularly the RNA as defined herein may comprise at least one heterologous 5′-UTR.

Preferably, the at least one 5′-UTR comprises or consists of a nucleic acid sequence derived from the 5′-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene, or from a variant of the 5′-UTR of a chordate gene, preferably a vertebrate gene, more preferably a mammalian gene, most preferably a human gene.

Preferably the artificial RNA of the present invention comprises a 5′-UTR, which may be derivable from a gene that relates to an RNA with an enhanced half-life (that provides a stable RNA), for example a 5′-UTR as defined and described below.

Preferably, the at least one heterologous 5′-UTR comprises a nucleic acid sequence derived from a 5′-UTR of a gene, which preferably encodes a stable mRNA, or from a homolog, a fragment or a variant of said gene.

In preferred embodiments of the first aspect, the artificial RNA of the invention comprises at least one heterologous 5′-UTR, wherein the at least one heterologous 5′-UTR comprises a nucleic acid sequence derived from a 5′-UTR of gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or from a homolog, a fragment or variant of any one of these genes.

RPL32-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR derived from a 5′-UTR of a gene encoding a 60S ribosomal protein L32, or a homolog, variant, fragment or derivative thereof, wherein said 5′-UTR preferably lacks the 5′TOP motif. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of a 60S ribosomal protein L32 (RPL32) gene, preferably from a vertebrate, more preferably a mammalian, most preferably a human 60S ribosomal protein L32 (RPL32) gene, or a homolog, variant, fragment or derivative thereof, wherein the 5′-UTR preferably does not comprise the 5′TOP of said gene. Said gene may preferably encode a 60S ribosomal protein L32 (RPL32) corresponding to a human 60S ribosomal protein L32 (RPL32) (UniProt Ref. No. P62899, entry version #138 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a RPL32 gene, wherein said 5′-UTR derived from a RPL32 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21 or 22 or a fragment or a variant thereof.

HSD17B4-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR derived from a 5′-UTR of a gene encoding a 17-beta-hydroxysteroid dehydrogenase 4, or a homolog, variant, fragment or derivative thereof, preferably lacking the 5′TOP motif. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of a 17-beta-hydroxysteroid dehydrogenase 4 (also referred to as peroxisomal multifunctional enzyme type 2) gene, preferably from a vertebrate, more preferably mammalian, most preferably human 17-beta-hydroxysteroid dehydrogenase 4 (HSD17B4) gene, or a homolog, variant, fragment or derivative thereof, wherein preferably the 5′-UTR does not comprise the 5′TOP of said gene. Said gene may preferably encode a 17-beta-hydroxysteroid dehydrogenase 4 protein corresponding to human 17-beta-hydroxysteroid dehydrogenase 4 (UniProt Ref. No. Q9BPX1, entry version #139 of Aug. 30, 2017), or a homolog, variant, fragment or derivative thereof. Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a HSD17B4 gene, wherein said 5′-UTR derived from a HSD17B4 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or 2 or a fragment or a variant thereof.

ASAH1-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR derived from a 5′-UTR of a gene encoding acid ceramidase (ASAH1), or a homolog, variant, fragment or derivative thereof. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of an acid ceramidase (ASAH1) gene, preferably from a vertebrate, more preferably mammalian, most preferably human acid ceramidase (ASAH1) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode an acid ceramidase (ASAH1) protein corresponding to human acid ceramidase (ASAH1) (UniProt Ref. No. Q13510, entry version #177 of Jun. 7, 2017), or a homolog, variant, fragment or derivative thereof. Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a ASAH1 gene, wherein said 5′-UTR derived from a ASAH1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or 4 or a fragment or a variant thereof.

ATP5A1-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR which is derived from a 5′-UTR of a gene encoding mitochondrial ATP synthase subunit alpha (ATP5A1), or a homolog, variant, fragment or derivative thereof, wherein said 5′-UTR preferably lacks the 5′TOP motif. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of a mitochondrial ATP synthase subunit alpha (ATP5A1) gene, preferably from a vertebrate, more preferably a mammalian and most preferably a human mitochondrial ATP synthase subunit alpha (ATP5A1) gene, or a homolog, variant, fragment or derivative thereof, wherein the 5′-UTR preferably does not comprise the 5′TOP of said gene. Said gene may preferably encode a mitochondrial ATP synthase subunit alpha protein corresponding to human acid mitochondrial ATP synthase subunit alpha (UniProt Ref. No. P25705, entry version #208 of Aug. 30, 2017), or a homolog, variant, fragment or derivative thereof. Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a ATP5A1 gene, wherein said 5′-UTR derived from a ATP5A1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5 or 6 or a fragment or a variant thereof.

MP68-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR which is derived from a 5′-UTR of a gene encoding MP68, or a homolog, fragment or variant thereof. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of a 6.8 kDa mitochondrial proteolipid (MP68) gene, preferably from a vertebrate, more preferably a mammalian 6.8 kDa mitochondrial proteolipid (MP68) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a 6.8 kDa mitochondrial proteolipid (MP68) protein corresponding to a human 6.8 kDa mitochondrial proteolipid (MP68) protein (UniProt Ref. No. P56378, entry version #127 of 15 Feb. 2017). Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a MP68 gene, wherein said 5′-UTR derived from a MP68 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or 8 or a fragment or a variant thereof.

NDUFA4-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR which is derived from a 5′-UTR of a gene encoding a Cytochrome c oxidase subunit (NDUFA4), or a homolog, fragment or variant thereof. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of a Cytochrome c oxidase subunit (NDUFA4) gene, preferably from a vertebrate, more preferably a mammalian Cytochrome c oxidase subunit (NDUFA4) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a Cytochrome c oxidase subunit (NDUFA4) protein corresponding to a human Cytochrome c oxidase subunit (NDUFA4) protein (UniProt Ref. No. 000483, entry version #149 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a NDUFA4 gene, wherein said 5′-UTR derived from a NDUFA4 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 or 10 or a fragment or a variant thereof.

NOSIP-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR which is derived from a 5-UTR of a gene encoding a Nitric oxide synthase-interacting (NOSIP) protein, or a homolog, variant, fragment or derivative thereof. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of a Nitric oxide synthase-interacting protein (NOSIP) gene, preferably from a vertebrate, more preferably a mammalian, most preferably a human Nitric oxide synthase-interacting protein (NOSIP) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a Nitric oxide synthase-interacting protein (NOSIP) protein corresponding to a human Nitric oxide synthase-interacting protein (NOSIP) protein (UniProt Ref. No. Q9Y314, entry version #130 of 7 Jun. 2017). Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a NOSIP gene, wherein said 5′-UTR derived from a NOSIP gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11 or 12 or a fragment or a variant thereof.

RPL31-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR which is derived from a 5′-UTR of a gene encoding a 60S ribosomal protein L31, or a homolog, variant, fragment or derivative thereof, wherein said 5′-UTR preferably lacks the 5′TOP motif. Such 5′-UTR preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of a 60S ribosomal protein L31 (RPL31) gene, preferably from a vertebrate, more preferably a mammalian60S ribosomal protein L31 (RPL31) gene, or a homolog, variant, fragment or derivative thereof, wherein the 5′-UTR preferably does not comprise the 5′TOP of said gene. Said gene may preferably encode a 60S ribosomal protein L31 (RPL31) corresponding to a human 60S ribosomal protein L31 (RPL31) (UniProt Ref. No. P62899, entry version #138 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a RPL31 gene, wherein said 5′-UTR derived from a RPL31 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or 14 or a fragment or a variant thereof.

SLC7A3-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR which is derived from a 5′-UTR of a gene encoding a cationic amino acid transporter 3 (solute carrier family 7 member 3, SLC7A3) protein, or a homolog, variant, fragment or derivative thereof. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of a cationic amino acid transporter 3 (SLC7A3) gene, preferably from a vertebrate, more preferably a mammalian cationic amino acid transporter 3 (SLC7A3) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a cationic amino acid transporter 3 (SLC7A3) protein corresponding to a human cationic amino acid transporter 3 (SLC7A3) protein (UniProt Ref. No. Q8WY07, entry version #139 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a SLC7A3 gene, wherein said 5′-UTR derived from a SLC7A3 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or 16 or a fragment or a variant thereof.

TUBB4B-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR which is derived from a 5′-UTR of a gene encoding a tubulin beta-4B chain (TUBB4B) protein, or a homolog, variant, fragment or derivative thereof. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of a tubulin beta-4B chain (TUBB4B) gene, preferably from a vertebrate, more preferably a mammalian and most preferably a human tubulin beta-48 chain (TUBB4B) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode a tubulin beta-4B chain (TUBB4B) protein corresponding to human tubulin beta-4B chain (TUBB4B) protein (UniProt Ref. No. Q8WY07, entry version #142 of 30 Aug. 2017). Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a TUBB4B gene, wherein said 5′-UTR derived from a TUBB4B gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17 or 18 or a fragment or a variant thereof.

UBQLN2-derived 5′-UTR: The artificial RNA of the invention may comprise a 5′-UTR which is derived from a 5′-UTR of a gene encoding an ubiquilin-2 (UBQLN2) protein, or a homolog, variant, fragment or derivative thereof. Such 5′-UTRs preferably comprise or consist of a nucleic acid sequence derived from the 5′-UTR of an ubiquilin-2 (UBQLN2) gene, preferably from a vertebrate, more preferably a mammalian ubiquilin-2 (UBQLN2) gene, or a homolog, variant, fragment or derivative thereof. Said gene may preferably encode an ubiquilin-2 (UBQLN2) protein corresponding to UniProt Ref. No. Q9UHD9, entry version #151 of 30 Aug. 2017. Accordingly, the artificial RNA of the invention may comprise a 5′-UTR derived from a UBQLN2 gene, wherein said 5′-UTR derived from a UBQLN2 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19 or 20 or a fragment or a variant thereof.

Further 5′-UTRs: In embodiments, the artificial RNA as defined herein comprises a 5′-UTR as described in WO2013/143700. In this context, the disclosure of WO2013/143700 relating to 5′-UTR sequences is herewith incorporated by reference. Particularly preferred 5′-UTRs are nucleic acid sequences derived from SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patent application WO2013/143700, or fragments or variants of these sequences. In this context, it is preferred that the 5′-UTR of the artificial RNA according to the present invention comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patent application WO2013/143700. In other embodiments, the artificial RNA of the invention comprises a 5′-UTR as described in WO2016/107877. In this context, the disclosure of WO2016/107877 relating to 5′-UTR sequences is herewith incorporated by reference. Particularly preferred 5′-UTRs are nucleic acid sequences according to SEQ ID NOs: 25 to 30 and SEQ ID NOs: 319 to 382 of the patent application WO2016/107877, or fragments or variants of these sequences. In this context, it is particularly preferred that the 5′-UTR of the artificial RNA comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according SEQ ID NOs: 25 to 30 and SEQ ID NOs: 319 to 382 of the patent application WO2016/107877. In other embodiments, the artificial RNA of the invention comprises a 5′-UTR as described in WO2017/036580. In this context, the disclosure of WO2017/036580 relating to 5′-UTR sequences is herewith incorporated by reference. Particularly preferred 5′-UTRs are nucleic acid sequences according to SEQ ID NOs: 1 to 151 of the patent application WO2017/036580, or fragments or variants of these sequences. In this context, it is particularly preferred that the 5′-UTR of the artificial RNA comprises or consists of a corresponding RNA sequence of the nucleic acid sequence according to SEQ ID NOs: 1 to 151 of the patent application WO2017/036580.

The inventors observed that certain combinations of at least one heterologous 5′-UTR and/or at least one heterologous 3′-UTR are advantageously increasing the translation of the at least one coding sequence operably linked to said 3′-UTR and/or 5′-UTR encoding at least one antigenic peptide or protein derived from a RSV F protein in the target tissue (e.g., muscle, dermis).

Accordingly it is preferred that the at least one heterologous 5′-UTR as defined herein and the at least one heterologous 3′-UTR as defined herein act synergistically to increase production (that is translation) of antigenic peptide or protein from the artificial RNA of the invention. These advantageous combinations of 5′-UTR and 3-UTR are specified in the following. Each of the abbreviation introduced below, namely “a-1”, “a-2”, “a-3”, “a-4”, “a-5”, “b-1”, “b-2”, “b-3”, “b-4”, “b-5”, “c-1”, “c-2”, “c-3” “c-4”, “c-5”, “d-1”, “d-2”, “d-3”, “d-4”, “d-5”, “e-1” “e-2” “e-3”, “e-4”, “e-5”, “e-6”, “f-1”, “f-2”, “f-3”, “f-4”, “f-5, “g-1”, “g-2”, “g-3”, “g-4”, “g-5”, “h-1”, “h-2”, “h-3”, “h-4”, “h-5”, i-1”, “i-2”, “i-3”, are used throughout the specification of the present invention and represent one advantageous combination of 5′-UTR and/or 3′UTR of the invention.

Accordingly, in preferred embodiments of the first aspect, the artificial RNA of the invention comprises

-   a-1. at least one 5′-UTR derived from a 5′-UTR of a HSD174 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   a-2. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   a-3. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   a-4. at least one 5′-UTR from a 5′-UTR of a NOSIP gene, or from a     corresponding RNA sequence, homolog, fragment or variant thereof and     at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a     corresponding RNA sequence, homolog, fragment or variant thereof; or -   a-5. at least one 5′-UTR derived from a 5′-UTR of a MP68 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   b-1. at least one 5′-UTR derived from a 5′-UTR of a UBQLN2 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3-UTR derived from a 3′-UTR of a RPS9 gene,     or from a corresponding RNA sequence, homolog, fragment or variant     thereof; or -   b-2. at least one 5′-UTR derived from a 5′-UTR of a ASAH1 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   b-3. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   b-4. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   b-5. at least one 5′-UTR derived from a 5′-UTR of a NOSIP gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   c-1. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   c-2. at least one 5′-UTR derived from a 5′-UTR of a NOSIP gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   c-3. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   c-4. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   c-5. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   d-1. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   d-2. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   d-3. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a GNAS     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   d-4. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   d-5. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   e-1. at least one 5′-UTR derived from a 5′-UTR of a TUBB4B gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   e-2. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   e-3. at least one 5′-UTR derived from a 5′-UTR of a MP68 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   e-4. at least one 5′-UTR derived from a 5′-UTR of a NOSIP gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   e-5. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   e-6. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   f-1. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a GNAS     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   f-2. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   f-3. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   f-4 at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3-UTR of a GNAS gene,     or from a corresponding RNA sequence, homolog, fragment or variant     thereof; or -   f-5. at least one 5′-UTR derived from a 5′-UTR of a MP68 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   g-1. at least one 5′-UTR derived from a 5′-UTR of a MP68 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   g-2. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   g-3. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a GNAS     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   g-4. at least one 5′-UTR derived from a 5′-UTR of a NOSIP gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   g-5. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   h-1. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   h-2. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a GNAS     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   h-3. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   h-4. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3-UTR derived from a 3′-UTR of a CASP1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   h-5. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof; or -   i-1. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof. -   i-2. at least one 5′-UTR derived from a 5′-UTR of a RPL32 gene, or     from a corresponding RNA sequence, homolog, fragment or variant     thereof and at least one 3′-UTR derived from a 3′-UTR of a ALB7     gene, or from a corresponding RNA sequence, homolog, fragment or     variant thereof. -   i-3. at least one 3′-UTR derived from a 3′-UTR of a alpha-globin     gene gene, or from a corresponding RNA sequence, homolog, fragment     or variant thereof.

Suitably, the artificial RNA of the invention comprises at least one coding sequence encoding at least one antigenic peptide or protein derived from a RSV F protein as specified herein operably linked to a 3′-UTR and a 5′-UTR selected from a-1 (HSD17B4/PSMB3), a-2 (Ndufa4/PSMB3), a-3 (SLC7A3/PSMB3), a-4 (NOSIP/PSMB3), a-5 (MP68/PSMB3), b-1 (UBQLN2/RPS9), b-2 (ASAH1/RPS9), b-3 (HSD17B4/RPS9), b-4 (HSD17B4/CASP1), b-5 (NOSIP/COX6B1), c-1 (NDUFA4/RPS9), c-2 (NOSIP/NDUFA1), c-3 (NDUFA4/COX6B1), c-4 (NDUFA4/NDUFA1), c-5 (ATP5A1/PSMB3), d-1 (Rpl31/PSMB3), d-2 (ATP5A1/CASP1), d-3 (SLC7A3/GNAS), d-4 (HSD17B4/NDUFA1), d-5 (Sc7a3/Ndufa1), e-1 (TUBB4B/RPS9), e-2 (RPL31/RPS9), e-3 (MP68/RPS9), e-4 (NOSIP/RPS9), e-5 (ATP5A1/RPS9), e-6 (ATP5A1/COX6B1), f-1 (ATP5A1/GNAS), f-2 (ATP5A1/NDUFA1), f-3 (HSD17B4/COX6B1), f-4 (HSD17B4/GNAS), f-5 (MP68/COX6B1), g-1 (MP68/NDUFA1), g-2 (NDUFA4/CASP1), g-3 (NDUFA4/GNAS), g-4 (NOSIP/CASP1), g-5 (RPL31/CASP1), h-1 (RPL31/COX6B1), h-2 (RPL31/GNAS), h-3 (RPL31/NDUFA1), h-4 (Slc7a3/CASP1), h-5 (SLC7A3/COX6B1), i-1 (SLC7A3/RPS9), i-2 (RPL32/ALB7), or i-3 (a-globin gene).

In particularly preferred embodiments of the first aspect, the artificial RNA of the invention comprise UTR elements according to a-1 (HSD17B4/PSMB3), a-4 (NDUFA4/PSMB3), c-1 (NDUFA4/RPS9), e-4 (NOSIP/RPS9), g-2 (NDUFA4/CASP1), i-2 (RPL32/ALB7), or i-3 (alpha-globin (muag)).

In a particularly preferred embodiment of the first aspect, the artificial RNA of the invention comprises UTR elements according to a-1 (HSD17B4/PSMB3).

The invention relates to an artificial RNA, preferably an RNA suitable for vaccination, comprising at least one heterologous 5′-UTR as defined above and/or at least one heterologous 3′-UTR as defined above and at least one coding sequence operably linked to said 3′-UTR and/or 5′-UTR, wherein said coding sequence encodes at least one antigenic peptide or protein derived from a Respiratory syncytial virus (“RSV”), or a fragment or variant thereof.

As used herein, the term “Respiratory syncytial virus” or the corresponding abbreviation “RSV” is not limited to a particular virus strain, variant, serotype, or isolate, etc. comprising any Respiratory syncytial virus of any origin.

According to various embodiments, the artificial RNA, preferably the coding sequence of the artificial RNA comprises or consists of a nucleic acid sequence that is derived from viruses, with NCBI Taxonomy ID (“NCBI-ID”) provided in List 1 below.

List 1: RSV Virus Strains:

Human orthopneumovirus, HRSV (NCBI-ID 11250); Human respiratory syncytial virus A, HRSV-A, Respiratory syncytial virus group A (NCBI-ID 208893); Human respiratory syncytial virus A strain Long, Human respiratory syncytial virus (subgroup A/strain Long) (NCBI-ID 11260); Human respiratory syncytial virus A2, Human respiratory syncytial virus (strain A2), HRSVA (NCBI-ID 11259); Human respiratory syncytial virus (strain RSB1734), (NCBI-ID 11253); Human respiratory syncytial virus (strain RSB5857) (NCBI-ID 11254); Human respiratory syncytial virus (strain RSB6190), (NCBI-ID 11255); Human respiratory syncytial virus (strain RSB6256), (NCBI-ID 11256); Human respiratory syncytial virus (strain RSB642), (NCBI-ID 11252); Human respiratory syncytial virus (strain RSB6614), (NCBI-ID 11257); Human respiratory syncytial virus B, HRSV-B, Respiratory syncytial virus group B, (NCBI-ID 208895); Human Respiratory syncytial virus 9320 (NCBI-ID 253182); Human respiratory syncytial virus B1 (NCBI-ID 79692); Human respiratory syncytial virus (subgroup B/strain 18537), (NCBI-ID 11251); Human respiratory syncytial virus (subgroup B/strain 8/60), (NCBI-ID 11258); Human respiratory syncytial virus S2, (NCBI-ID 410078); Human respiratory syncytial virus strain RSS-2, (NCBI-ID 11261); unclassified Human respiratory syncytial virus, (NCBI-ID 410233); Human respiratory syncytial virus (strain RSP112/Sweden/02-03), (NCBI-ID 410237); Human respiratory syncytial virus (strain RSP120/Sweden/02-03), (NCBI-ID 410238); Human respiratory syncytial virus (strain RSP121/Sweden/02-03), (NCBI-ID 410239); Human respiratory syncytial virus (strain RSP122/Sweden/02-03), (NCBI-ID 410247); Human respiratory syncytial virus (strain RSP13/Sweden/02-03), (NCBI-ID 410241); Human respiratory syncytial virus (strain RSP140/Sweden/02-03), (NCBI-ID 410248); Human respiratory syncytial virus (strain RSP16/Sweden/02-03), (NCBI-ID 410242); Human respiratory syncytial virus (strain RSP171/Sweden/02-03), (NCBI-ID 410246); Human respiratory syncytial virus (strain RSP183/Sweden/02-03), (NCBI-ID 410249); Human respiratory syncytial virus (strain RSP191/Sweden/02-03), (NCBI-ID 410240); Human respiratory syncytial virus (strain RSP199/Sweden/02-03), (NCBI-ID 410250); Human respiratory syncytial virus (strain RSP212/Sweden/02-03), (NCBI-ID 410251); Human respiratory syncytial virus (strain RSP41/Sweden/02-03), (NCBI-ID 410234); Human respiratory syncytial virus (strain RSP45/Sweden/02-03), (NCBI-ID 410235); Human respiratory syncytial virus (strain RSP56/Sweden/02-03), (NCBI-ID 410243); Human respiratory syncytial virus (strain RSP58/Sweden/02-03), (NCBI-ID 410236); Human respiratory syncytial virus (strain RSP67/Sweden/02-03), (NCBI-ID 410244); Human respiratory syncytial virus (strain RSP94/Sweden/02-03) (NCBI-ID 410245); Respiratory syncytial virus isolate RSV Memphis-37, (strain Memphis-37) (NCBI-ID 12814).

In preferred embodiments of the invention, the at least one antigenic peptide or protein is derived from a Respiratory syncytial virus isolate RSV Memphis-37 (strain Memphis-37) (NCBI-ID: 12814). Throughout the present invention, including the information contained in the ST25 sequence listing, the abbreviation “HRSV(Memphis-37)” is used for said particularly preferred RSV virus.

In preferred embodiments of the invention, the at least one antigenic peptide or protein is derived from a Human respiratory syncytial virus A2, Human respiratory syncytial virus (strain A2) (NCBI-ID: 11259). Throughout the present invention, including the information contained in the ST25 sequence listing, the abbreviation “HRSV(A2)” is used for said particularly preferred RSV virus.

It has to be understood that the skilled person may also use amino acid sequences and nucleic acid sequences derived from any RSV strains provided in List 1 to adapt the teaching of the present invention and to obtain RNA constructs, compositions, and vaccines according to the invention.

In various embodiments, the at least one antigenic peptide or protein may be selected from fusion protein (F), glycoprotein G, short hydrophobic protein SH, matrix protein M, nucleoprotein N, large polymerase L, M2-1 protein, M2-2 protein, phosphoprotein P, non-structural protein NS1, or non-structural protein NS2 of Respiratory syncytial virus (RSV) or a fragment, variant or derivative thereof.

In particularly preferred embodiments of the first aspect, the at least one antigenic peptide or protein is derived from an RSV fusion (F) protein. In this context, the amino acid sequence of the at least one antigenic peptide or protein may be selected from any peptide or protein derived from RSV fusion protein F or from a fragment, variant or derivative thereof.

RSV F protein is initially expressed (after infection of a host cell) as a single polypeptide precursor, designated full-length fusion protein F (herein referred to as “F0”). F0 forms a trimer in the endoplasmic reticulum and is processed by a cellular/host furin-like protease at two conserved sites, generating, F1, F2, and Pep27 polypeptides. The Pep27 polypeptide is excised and does not form part of the mature F protein. The F2 polypeptide originates from the N-terminal portion of the F0 precursor and links to the F1 polypeptide via two disulfide bonds. The F1 polypeptide originates from the C-terminal portion of the F0 precursor and anchors the mature F protein in the membrane via a transmembrane domain, which is linked to a cytoplasmic tail. Three F2-F1 heterodimer units (“protomers”) assemble to form a mature F protein. Initially, the mature F protein is in a metastable form (herein referred to as “pre-fusion conformation”). Upon triggering, it undergoes a dramatic and irreversible conformational change (herein referred to as “postfusion conformation”) that fuses the viral and target-cell membranes.

Accordingly, the artificial RNA of the first aspect, preferably the artificial RNA suitable for vaccination, encodes at least one antigenic peptide or protein derived from a RSV F protein or a fragment or variant thereof.

In preferred embodiments, at least one antigenic peptide or protein derived from a RSV F protein may be derived from any one of the following amino acid sequences (NCBI Protein Accession numbers) provided in List 2 below.

List 2: NCBI Protein Accession Numbers of RSV Fusion (F) Proteins:

Accession No. Protein, AJF44801.1, AJF44759.1, AJF44661.1, AJF44602.1, 2207424A, AAB38520.1, AAB38517.1, AAB38519.1, AAB38518.1, AVQ93587.1, AVQ93599.1, AVQ93571.1, AVQ93568.1, AVQ93589.1, AVQ93597.1, AVQ93563.1, AVQ93594.1, AVQ93606.1, AVQ93601.1, AVQ93562.1, AVQ93561.1, AVQ93607.1, AVQ93588.1, AVQ93575.1, AVQ93468.1, AVQ93467.1, AVQ93590.1, AVQ93552.1, AVQ93556.1, AVQ93471.1, AVQ93458.1, AVQ93494.1, AVQ93470.1, AVQ93489.1, AVQ93542.1, AVQ93472.1, AVQ93514.1, AVQ93485.1, AVQ93533.1, AVQ93481.1, AVQ93546.1, AVQ93512.1, AVQ93554.1, AVQ93551.1, AVQ93547.1, AVQ93558.1, AVQ93461.1, AVQ93500.1, AVQ93426.1, AVQ93398.1, AVQ93401.1, AVQ93361.1, AVQ93408.1, AVQ93443.1, AVQ93429.1, AVQ93359.1, AVQ93365.1, AVQ93366.1, AVQ93402.1, AVQ93377.1, AVQ93412.1, AVQ93391.1, AVQ93457.1, AVQ93372.1, AVQ93455.1, AVQ93364.1, AVQ93378.1, AVQ93393.1, AVQ93362.1, AVQ93585.1, ART28504.1, AVQ93404.1, AOS49123.1, AOS48496.1, AMT78271.1, AHX57174.1, AHW81390.1, AHV81506.1, AFX60128.1, AFX60129.1, AEQ63389.1, ARB66328.1, ANZ80034.1, AMN91253.1, P03420.1, A1008046.1, NP_056863.1, AFX60234.1, AFX60231.1, AFX60232.1, AFX60222.1, AFX60219.1, AFX60215.1, AFX60214.1, AFX60212.1, AFX60208.1, AFX60202.1, AFX60220.1, AFX60213.1, AFX60190.1, AFX60187.1, AFX60201.1, AFX60173.1, AFX60169.1, AFX60162.1, AFX60156.1, AFX60151.1, AFX60150.1, AFX60148.1, AFX60141.1, AFX60127.1, AFX60137.1, AFX60135.1, AFV46420.1, AFX60200.1, AFV46419.1, AFV46417.1, AFV46413.1, AFV46414.1, AFV46410.1, AFV46403.1, AFV46409.1, AFP99061.1, AFM95400.1, AFV46401.1, AFP99064.1, AFM95376.1, AFX60138.1, AFP99060.1, AFM95365.1, AFM55563.1, AFM55530.1, AFM55442.1, AFM55420.1, AFM55552.1, AFM55365.1, AFP99059.1, AFM95385.1, AFM55354.1, AFM55343.1, AFM55387.1, AFM55299.1, AFM55288.1, AFM55266.1, AFM55277.1, AFM55255.1, AFM55222.1, AFM55211.1, AFI25262.1, AFD34266.1, AFM55332.1, AFD34264.1, AFD34262.1, AFD34265.1, AFD34261.1, AFD34260.1, AFD34259.1, AEQ63641.1, AEQ63487.1, AEQ63520.1, AEQ63378.1, AEQ63367.1, 4CCF_A, AEQ63334.1, AEO45949.1, AEO45939.1, AEQ63312.1, AEQ63586.1, AEO45919.1, AEO45909.1, AEO45889.1, AEO45879.1, AEO45869.1, AEO45929.1, AEO45850.1, AEO45859.1, AEQ63444.1, AEO23054.1, AEO23052.1, AEO23051.1, AEC32087.1, ADZ95785.1, AEC32085.1, ADZ95784.1, ADZ95783.1, ADZ95782.1, ADZ95781.1, ADZ95779.1, ADZ95780.1, ADZ95777.1, ADZ95778.1, ADZ95776.1, ADZ95775.1, ACY68435.1, ACO83302.1, AB35685.1, AFI25251.1, AAX23994.1, AAQ97026.1, AAR14266.1, AAQ97027.1, AAQ97030.1, AAQ97028.1, AAC57027.1, AAQ97029.1, AAQ97031.1, AAM68160.1, AAM44851.1, P11209.2, P13843.1, AAO72325.1, AAM68157.1, CAA26143.1, 1512372A, AAB86664.1, AAO72324.1, AAB82446.1, AAO72323.1, AAM68154.1, AAA47410.1, P12568.1, ARB07894.1, AGG39517.1, BBC54612.1, BBC54636.1, BBC54627.1, BBC54621.1, BBC54570.1, BBC54579.1, BBC54595.1, BBC54555.1, BBC54552.1, BBC54564.1, BBC54581.1, BBC54571.1, BBC54553.1, BBC54565.1, BBC54245.1, BBC54243.1, BBC54238.1, BBC54242.1, BBC54239.1, BBC54235.1, BBC54234.1, BBC54236.1, BBC54244.1, BBC54186.1, BBC54178.1, BBC54202.1, BBC54151.1, BBC54142.1, BBC54134.1, BBC54170.1, BBC54210.1, BBC54169.1, BBC54213.1, BBC54203.1, BBC54160.1, BBC54163.1, BBC54215.1, BBC54156.1, BBC54179.1, BBC54150.1, BBC54207.1, BBC54194.1, BBC54149.1, BBC54138.1, BBC54199.1, BBC54220.1, BBC54181.1, BBC54132.1, BBC54146.1, BBC54122.1, BBC54124.1, BBB35202.1, BBB35201.1, BBB35192.1, BBB35193.1, BBB35199.1, BBB35184.1, BBB35126.1, BBB35133.1, BBB35130.1, BBB35160.1, BBB35162.1, BBB35181.1, BBB35165.1, BBB35121.1, BBB35138.1, BBB35176.1, BBB35142.1, BBB35136.1, BBB35153.1, BBB35115.1, BBB35150.1, BBB35097.1, BBB35109.1, BBB35094.1, BBB35183.1, BBB35104.1, BBB35099.1, BBB35188.1, AKA45871.1, ASV65838.1, AGG39373.1, AII22107.1, AGG39400.1, AGG39457.1, ARR29240.1, ARR29251.1, ARR29189.1, ARR29207.1, AUH15164.1, ATV81343.1, AUC68654.1, AUC68577.1, AUC68566.1, AUC68555.1, AUC68478.1, AUC68445.1, AUC68500.1, AUC68522.1, AUC68291.1, AUC68149.1, AUC68094.1, AMA67097.1, AMA66866.1, AMA66580.1, AIZ95750.1, AIZ95541.1, AIZ95519.1, AHY21419.1, AHY21331.1, AHY21165.1, AHY21143.1, AHY21132.1, AGG39478.1, ATV93506.1, ATV93509.1, AIZ95717.1, ATV81354.1, ART28317.1, BBA57890.1, BBA57901.1, ASZ70099.1, ART28361.1, AQX36844.1, ASK05520.1, ART28427.1, ART28339.1, ART28297.1, ART28328.1, ARQ15966.1, ARN61507.1, ARA15413.1, AGG39394.1, APW78845.1, APW78867.1, APW78900.1, APW78878.1, APW78779.1, APW78702.1, APW78713.1, APW78680.1, APW78658.1, APW78647.1, APW78636.1, AOS48870.1, APW78614.1, AMT78905.1, AMT77402.1, AOZ15479.1, AGN28484.1, AHX57240.1, AHX57031.1, AOS48980.1, AOS48848.1, AOS48815.1, AOS48738.1, AOS49068.1, AOS48727.1, AOS48716.1, AOS48683.1, AOS48551.1, AOS48485.1, AOS48518.1, AOS48441.1, AOS48397.1, AOS48375.1, AOS48353.1, AOS48287.1, AOS48320.1, AOS48265.1, AOS48254.1, AOS48221.1, ANZ79638.1, ALC74025.1, AHV81286.1, AOD40888.1, AOD40516.1, AOD40214.1, AOD40125.1, AOD40104.1, AOD40082.1, AOD39908.1, AJF44826.1, AJF44506.1, AJF44535.1, ANZ80463.1, ANZ80408.1, ANZ80397.1, ANZ80386.1, ANZ80331.1, ANZ80320.1, ANZ80364.1, ANZ80276.1, ANZ80221.1, ANZ80188.1, ANZ80144.1, ANZ80133.1, ANZ80111.1, ANZ80122.1, ANZ80056.1, ANZ80012.1, ANZ80067.1, ANZ79979.1, ANZ79935.1, ANZ79990.1, ANZ79902.1, ANZ79759.1, ANZ79715.1, ANZ79671.1, AMT79718.1, AMT79586.1, AMT79553.1, AMT79542.1, AMT79476.1, AMT79388.1, AMT79201.1, AMT79190.1, AMT79157.1, AMT79124.1, AMT79091.1, AMT79047.1, AMT79014.1, AMT79003.1, AMT78872.1, AMT78832.1, AMT78689.1, AMT78645.1, AMT78513.1, AMT78480.1, AMT78447.1, AMT78392.1, AMT78194.1, AMT78084.1, AMT78051.1, AMT77963.1, AMT77908.1, AMT77776.1, AMT77710.1, AMT77424.1, AMN91385.1, AMN91264.1, AMN91242.1, AHX57504.1, CUS01881.1, CUS01880.1, CUS01877.1, CUS01874.1, CUS01870.1, CUS01875.1, AMA67350.1, AMA67251.1, AMA67262.1, AMA67229.1, AMA67196.1, AMA67130.1, AMA67185.1, AMA67163.1, AMA67086.1, AMA67075.1, AMA66998.1, AMA66987.1, AMA66976.1, AMA66965.1, AMA66921.1, AMA66844.1, AMA66833.1, AMA66624.1, AMA66613.1, AMA66591.1, AMA66569.1, AMA66547.1, AMA66503.1, AMA66492.1, AMA66481.1, AMA66448.1, AMA66415.1, AMA66393.1, AMA66360.1, AGG39553.1, AGG39469.1, AJZ70144.1, AJZ70166.1, AJZ70155.1, AJZ70133.1, AJZ70067.1, AJZ70001.1, AJZ69990.1, AJZ69968.1, AJZ69946.1, AJZ69913.1, AJZ69880.1, AJZ69847.1, AJZ69869.1, AJZ69770.1, AJZ69748.1, AJZ69726.1, AJZ69682.1, AJZ69671.1, AJZ69704.1, AJZ69660.1, AJZ69715.1, AJZ69627.1, AJZ69616.1, AJZ69638.1, AJO16077.1, AJO16055.1, AKE31881.1, AKE31882.1, AKE31878.1, AJF44835.1, AJF44790.1, AJF44737.1, AJF44716.1, AJF44725.1, AJF44643.1, AJF44628.1, AJF44624.1, AJF44613.1, AJF44566.1, AJF44555.1, AJF44526.1, AJF44517.1, AKA45882.1, AKA45849.1, AHA83913.1, AGN28715.1, AHA83902.1, AHA83837.1, AHA83826.1, AHA83705.1, AHA83630.1, AGG39505.1, AIY70242.1, AIY70198.1, AII30203.1, AHY21463.1, AHY21397.1, AHY21320.1, AHY21298.1, AHY21287.1, AHY21276.1, AHY21254.1, AHY21199.1, AHY21176.1, AHX57537.1, AHX57471.1, AHX57427.1, AHX57042.1, AIZ95981.1, AIZ95893.1, AIZ95871.1, AIZ95816.1, AIZ95772.1, AIZ95629.1, AIZ95673.1, AIZ95596.1, AIZ95585.1, AIZ95552.1, AEQ63553.2, AEQ63542.2, AEQ63575.1, AEQ63531.1, AEQ63498.1, AEQ63422.1, AEQ63411.1, AHX57570.1, AHX57152.1, AHX57064.1, AHX57009.1, AHX56987.1, AHV82100.1, AHV82001.1, AHV81891.1, AHV81880.1, AHV81836.1, AHV81682.1, AHV81649.1, AHV81484.1, AHV81462.1, AHV81385.1, AHV81363.1, AHV81330.1, AHV81253.1, AHV81154.1, AHV81122.1, AHV81089.1, AHV81012.1, AHV80957.1, AHV80880.1, AHV80869.1, AHV80836.1, AHV80803.1, AGG39559.1, AGG39562.1, AGG39556.1, AGG39550.1, AGG39547.1, AGG39544.1, AGG39541.1, AGG39529.1, AGG39526.1, AGG39523.1, AGG39514.1, AGG39502.1, AGG39499.1, AGG39496.1, AGG39493.1, AGG39484.1, AGG39490.1, AGG39487.1, AGG39475.1, AGG39472.1, AGG39466.1, AGG39463.1, AGG39454.1, AGG39442.1, AGG39439.1, AGG39436.1, AGG39415.1, AGG39403.1, AGG39397.1, AGG39391.1, AGG39379.1, AHL84194.1, AHA84012.1, AHJ60043.1, BAO49770.1, BAO49766.1, BAO49767.1, AHA84034.1, AHA84023.1, AHA83990.1, AHA83957.1, AHA83924.1, AHA83891.1, AHA83880.1, AHA83782.1, AHA83760.1, AHA83694.1, AGT75357.1, AGN92848.1, AGN28792.1, AGN28781.1, AGN28759.1, AGN28748.1, AGN28693.1, AGN28638.1, AGN28627.1, AGN28539.1, AGN28528.1, AGN28462.1, AGN28440.1, AGL96787.1, AGL96786.1, AGL96784.1, AAS93662.1, AAS93657.1, AAS93656.1, AAS93659.1, AAS93660.1, AAS93663.1, AAS93664.1, AAS93655.1, CUS01869.1, AHG54517.1, ASF87348.1, ASF87341.1, ASF87344.1, ASF87351.1, ASF87338.1, ASF87342.1, ASF87352.1, ASF87337.1, ASF87336.1, AEQ98756.1, AEQ98757.1, AEQ98755.1, AEQ98752.1, AEQ98753.1, AEQ98747.1, ASF87325.1, ASF87326.1, 5W23_A, 5EA3_F, 5UDD_A, 5EA8_F, AHG54458.1, AEN74947.1, AHG54485.1, AHG54477.1, AHG54451.1, AHG54463.1, AHG54445.1, AEO12131.1, AEN74945.1, AEN74944.1, ASF87335.1, AHG54515.1, AHA61605.1, AHV81660.1, AHG54441.1 or AIY60640.1.

In the context of the present invention, “RSV F protein”, “RSV fusion protein (F)”, “RSV F”, or “F” may be understood in its broadest sense and refers to F0 (F polypeptide precursor), F1, F2 and Pep27 polypeptides, F2-F1 heterodimer, or the mature F protein (comprising three F2-F1 heterodimers), or fragments and variants of any of these. Accordingly, the term “peptide or protein derived from a RSV fusion (F) protein” refers to a peptide, protein, fragment or variant derived from e.g. F0 (F protein polypeptide precursor), F1, F2 and Pep27 polypeptides, F2-F1 heterodimer, or the mature F protein. Additionally, the term “peptide or protein derived from a RSV fusion (F) protein” refers to peptide, protein, fragment or variant derived from “RSV F protein” or “RSV fusion protein (F)” as defined above which may be genetically engineered to e.g. to lack certain protein elements (e.g. the cytoplasmic tail, the furin cleavage site, Pep27) or e.g. comprise additional elements (e.g., linker elements, heterologous signal peptides etc.). For example, the term “peptide or protein derived from a RSV fusion (F) protein” refers to peptide, protein, fragment or variant derived from F0, F-del, F0_DSCav1, F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3, F_DSCav1_mut4, F_DSCav1_mut5, F_DSCav1_mut6, F_DSCav1_mut7, F_DSCav1_mut8, F_DSCav1_mut0, F-del_DSCav1, F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut4, F-del_DSCav1_mut5, F-del_DSCav1_mut6, F-del_DSCav1_mut7, F-del_DSCav1_mut8, F-del_DSCav1_mut0 (for explanation of the constructs see Table 1). Particularly suitable F protein variants that may be encoded by the RNA of the first aspect are specified in the following and are provided in Table 1.

It has to be noted that where reference is made to amino acid (aa) residues and their position in an RSV F protein, any numbering used herein—unless stated otherwise—relates to the position of the respective aa residue in a corresponding F0 precursor protein of HRSV(A2) (SEQ ID NO: 68) or a corresponding F0 precursor protein of HRSV(Memphis-37) (SEQ ID NO: 8937 or 11726) wherein position “1” corresponds to the first aa residue, i.e. the aa residue at the N-terminus of a HRSV(A2) F0 precursor protein or a HRSV(Memphis-37) F0 precursor protein.

In preferred embodiments, the at least one coding sequence of the RNA of the first aspect encodes at least one antigenic peptide or protein from RSV F protein, wherein RSV F protein is a full-length F protein (F0) or an F protein with deleted C-terminus (F-del), or a fragment or a variant thereof.

In this context, any RSV F full-length protein (precursor protein, referred to as “F0”) may be used as suitable antigen and may preferably be derived from any NCBI Protein Accession numbers provided in List 2 or may be chosen from any one of SEQ ID NOs: 68, 8279-8967 or 11726. In preferred embodiments of the invention, the full-length F protein (F0) of HRSV(A2) (SEQ ID NO: 68) is suitably used, see overview Table 1. In other preferred embodiments of the invention, the full-length F protein (F0) of HRSV(Memphis-37) (SEQ ID NO: 8937 or 11726) is suitably used, see overview Table 1.

In this context, any RSV F with deleted C-terminus (F-del) may be used as suitable antigen and may preferably be derived from any NCBI Protein Accession numbers provided in List 2 or may be chosen from any one of SEQ ID NOs: 483, 8968-9683, or 12095. An example of such a deletion mutant is the RSV-Fdel554-574 protein according to (Oomens et al. 2006. J. Virol. 80(21):10465-77) where aa residue 554-574 of the full-length F0 protein are removed. Deletion of the main part of the cytoplasmic tail (aa 554-574) of F0 leads to improved intracellular trafficking/cell surface transport in vitro and increases cell surface expression of RSV F significantly. Increased cell surface presentation results in improved B cell recognition (in line with published data; see WO2015024668). In preferred embodiments of the invention, F protein with deleted C-terminus, herein referred to as “F-del” (SEQ ID NO: 483, 9653 or 12095) is suitably used, see overview Table 1. In the light of high level of structural conservation of the RSV F protein among different RSV strains (see e.g. Hause et al., 2017, PLOS ONE 12(6): e0180623), the deletion of aa 554-574 is applicable to different RSV F protein sequences of different RSV isolates.

In particularly preferred embodiments, the artificial RNA of the first aspect encodes least one antigenic peptide or protein derived from RSV F protein, wherein said RSV F protein is designed to stabilize the antigen in pre-fusion conformation. A pre-fusion conformation is particularly advantageous in the context of an efficient RSV vaccine, as several potential epitopes for neutralizing antibodies are merely accessible in said protein conformation.

In several embodiments, the RSV F protein includes one or more amino acid substitutions that stabilize the F protein in the pre-fusion conformation, for example, substitutions that stabilize the membrane distal portion of the F protein (including the N-terminal region of the F1 polypeptide) in the pre-fusion conformation. For example, the amino acid substitution can introduce a non-natural disulfide bond or can be a cavity-filling amino acid substitution.

Accordingly, in several embodiments, a preferred RSV F protein includes S155C and S290C substitutions that form a non-natural disulfide bond that stabilizes the protein in a pre-fusion conformation; that is, in a conformation that specifically binds to one or more pre-fusion specification antibodies, and/or presents a suitable antigenic site that is present on the pre-fusion conformation but not in the postfusion conformation of RSV F protein. In further embodiments, the recombinant RSV F protein can additionally include F, L, W, Y, H, or M substitution at position 190, position 207, or positions 190 and 207.

An example of an RSV F protein designed to stabilize the antigen in pre-fusion conformation is RSV F protein comprising a DSCav1 mutation (S155C, S290C, S190F, and V207L), or a fragment or a variant thereof. Such RSV F DSCav1 proteins have been described in the art (W2014160463).

Accordingly, in particularly preferred embodiments, the artificial RNA of the first aspect encodes least one antigenic peptide or protein derived from RSV F protein, wherein the RSV F protein comprises a DSCav1 mutation (S155C, S290C, S190F, and V207L), or a fragment or a variant thereof.

It has to be understood in the context of the invention that any RSV F may be mutated at positions S155C, S290C, S190F, and V207L to stabilize the protein in the pre-fusion conformation and may be suitably used in the context of the invention. Accordingly, any NCBI Protein Accession numbers provided above, or any protein selected from SEQ ID NOs: 68, 8279-8967, 483, 8968-9683, 12095, or 11726 or fragments or variants thereof can be chosen by the skilled person to introduce such amino acid changes according to S155C, S290C, S190F, and V207L to generate various RSV F DSCav1 proteins.

In preferred embodiments, RSV F full-length protein (precursor protein, “F0”) of HRSV(A2) (SEQ ID NO: 68) is used to introduce S155C, S290C, S190F, and V207L amino acid changes, leading to an amino acid sequence according to SEQ ID NO: 898. Such a RSV F protein is herein referred to as “F0_DSCav1” throughout the present invention (see overview Table 1 (preferred RSV F protein antigen designs)).

In other preferred embodiments, RSV F_del protein (deleted cytoplasmic tail (aa 554-574)) of HRSV(A2) (SEQ ID NO: 483) is used to introduce S155C, S290C, S190F, and V207L amino acid changes, leading to an amino acid sequence according to SEQ ID NO: 1267. Such a RSV F protein is herein referred to as “F-del_DSCav1” throughout the present invention (see overview Table 1 (preferred RSV F protein antigen designs)).

In preferred embodiments, RSV F full-length protein (precursor protein, “F0”) of HRSV(Memphis-37) (SEQ ID NO: 8937 or 11726) is used to introduce S155C, S290C, S190F, and V207L amino acid changes, leading to an amino acid sequence according to SEQ ID NO: 12464. Such a RSV F protein is herein referred to as “FO_DSCav1” throughout the present invention (see overview Table 1 (preferred RSV F protein antigen designs)).

In other preferred embodiments, RSV F_del protein (deleted cytoplasmic tail (aa 554-574)) of HRSV(Memphis-37) (SEQ ID NO: 9653 or 12095) is used to introduce S155C, S290C, S190F, and V207L amino acid changes, leading to an amino acid sequence according to SEQ ID NO: 12833. Such a RSV F protein is herein referred to as “F-del_DSCav1” throughout the present invention (see overview Table 1 (preferred RSV F protein antigen designs).

In preferred embodiments, the at least one antigenic peptide or protein may be an engineered protein comprising the two subunits, F1 and F2 of mature F as a single polypeptide chain, wherein F2 and F1 are preferably connected via a linker (GS). Examples of said engineered F2-linker-F1 fusion proteins (e.g., F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553)) have been described in the art (Joyce, M. Gordon, et al. “Iterative structure-based improvement of a fusion-glycoprotein vaccine against RSV.” Nature structural & molecular biology 23.9 (2016): 811; WO2017172890). Said F2-linker-F1 RSV F proteins lack aa104-144 (comprising the furine cleavage site and Pep27) and comprise a linker element between F2 polypeptide and F1 polypeptide (e.g. GSlinker). Said F2-linker-F1 proteins may show superior properties in terms of stability and/or antigenicity.

Accordingly, in preferred embodiments, the RSV F protein comprises the two subunits F2 and F1 fused into a single polypeptide chain, wherein F2 and F1 are connected via a linker element, preferably a GS linker as specified herein, preferably generating a stable F2-linker-F1 proteins.

Preferably, said F2-linker-F1 fusion proteins, e.g. F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) additionally comprise a DScav1 mutation as outlined above (herein referred to as “mut0”; e.g., SEQ ID NOs: 3850, 4219 or 13940, 14309).

In particularly preferred embodiments, the RSV F protein may additionally comprise at least one further mutation selected from (S46G, A149C, S215P, Y458C, K465Q), (S46G, E92D, A149C, S215P, Y458C, K465Q), (S46G, N671, E92D, A149C, S215P, Y458C, K465Q), (A149C, Y458C), (N183GC, N428C), (Q98C, Q361C, S46G, E92D, L95M, S215P, I217P, I221M, R429K, K465Q), (Q98C, Q361C, L95M, I221M, R429K), or (N183GC, N428C, S46G, N671, E92D, S215P, K465Q) or a fragment or a variant thereof.

In particularly preferred embodiments, said F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553)) may additionally comprise, preferably in addition to the DSCav1 mutation, at least one mutation selected from S46G, A149C, S215P, Y458C, K465Q, herein referred to as “mut1”, e.g., SEQ ID NOs: 1636, 2005, or 14678, 15047; or S46G, E92D, A149C, S215P, Y458C, K465Q, herein referred to as “mut2”, e.g., SEQ ID NOs: 2374, 2743 or 15416, 15785; or S46G, N671, E92D, A149C, S215P, Y458C, K465Q, herein referred to as “mut3”, e.g., SEQ ID NOs: 3112, 3481 or 13202, 13571, or a fragment or a variant of any of these (see overview Table 1 (preferred RSV F protein antigen designs)).

In other embodiments, said F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553)) may additionally comprise, preferably in addition to the DSCav1 mutation, at least one mutation selected from A149C, Y458C, herein referred to as “mut4”, e.g., SEQ ID NOs: 4588, 4957 or 16154, 16523; or N183GC, N428C, herein referred to as “mut5”, e.g., SEQ ID NOs: 5326, 5695 or 16892, 17261; or Q98C, Q361C, S46G, E92D, L95M, S215P, I217P, I221M, R429K, K465Q, herein referred to as “mut6”, e.g., SEQ ID NOs: 6064, 6433 or 17630, 17999; or Q98C, Q361C, L95M, I221M, R429K, herein referred to as “mut7”, e.g., SEQ ID NOs: 6802, 7171 or 18368, 18737; or N183GC, N428C, S46G, N671, E92D, S215P, K465Q, herein referred to as “mut8”, e.g., SEQ ID NOs: 7540, 7909 or 19106, 19475, or a fragment or a variant thereof.

It has to be understood in the context of the invention that any RSV F may be adapted in a way that the two subunits, F1 and F2 are comprised in a single polypeptide chain, wherein F2 and F1 may be connected via a linker, e.g. a (GS) linker to enhance stability of the protein as explained for mutations “mut1”, “mut2” and “mut3”, e.g. by deleting aa104-144 of the F0 polypeptide chain (as explained above), by introducing a linker element between F2 and F1 as explained above, and by introducing suitable amino acid substitutions as explained above. Accordingly, any NCBI Protein Accession numbers provided above (see List 2), or any protein selected from SEQ ID NOs: 68, 8279-8967, 483, 8968-9683, 11726, 12095 or fragments or variants thereof can be adapted by the skilled person to generate F2-linker-F1 fusion proteins as outlined herein, and may additionally be adapted by introducing (S46G, A149C, S215P, Y458C, K465Q), (S46G, E92D, A149C, S215P, Y458C, K465Q), (S46G, N671, E92D, A149C, S215P, Y458C, K465Q), (A149C, Y458C), (N183GC, N428C), (Q98C, Q361C, S46G, E92D, L95M, S215P, I217P, I221M, R429K, K465Q), (Q98C, Q361C, L95M, I221M, R429K), or (N183GC, N428C, S46G, N671, E92D, S215P, K465Q) aa substitutions and/or a DSCav1 mutation. Moreover, apart from using a GS linker as outlined above, the skilled person may of course choose between various known linker elements to arrive at similar likewise suitable RSV F protein variants (e.g. selected from SEQ ID NOs: 117-162 of the patent application WO2017/172890 or fragments or variants of these sequences, or selected from SEQ ID NOs: 1509-1565 of the patent application WO2017/081082, or fragments or variants of these sequences).

In preferred embodiments, F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined above, herein referred to as F_DSCav1_mut0 or F-del_DSCav1_mut0. In preferred embodiments, F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined above, and additionally an amino acid substitution mut1 as defined above, herein referred to as F_DSCav1_mut1 or F-del_DSCav1_mut1. In preferred embodiments, F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined above, and additionally an amino acid substitution mut2 as defined above, herein referred to as F_DSCav1_mut2 or F-del_DSCav1_mut2. In preferred embodiments, F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined above, and additionally an amino acid substitution mut3 as defined above, herein referred to as F_DSCav1_mut3 or F-del_DSCav1_mut3. In preferred embodiments, F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined above, and additionally an amino acid substitution mut4 as defined above, herein referred to as F_DSCav1_mut4 or F-del_DSCav1_mut4. In preferred embodiments, F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined above, and additionally an amino acid substitution mut5 as defined above, herein referred to as F_DSCav1_mut5 or F-del_DSCav1_mut5. In preferred embodiments, F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined above, and additionally an amino acid substitution mut6 as defined above, herein referred to as F_DSCav1_mut6 or F-del_DSCav1_mut6 . In preferred embodiments, F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined above, and additionally an amino acid substitution mut7 as defined above, herein referred to as F_DSCav1_mut7 or F-del_DSCav1_mut7. In preferred embodiments, F2-linker-F1 proteins (F(1-103)-GS-F(145-574) or F(1-103)-GS-F(145-553) comprise a DSCav-1 mutation as outlined above, and additionally an amino acid substitution mut8 as defined above, herein referred to as F_DSCav1 mut8 or F-del_DSCav1 mut8.

A detailed description of particularly preferred RSV F proteins is provided in Table 1.

In Table 1 all references made to amino acid (aa) residues and their position in an RSV F protein relates to the position of the respective aa residue in a corresponding F0 precursor protein of HRSV(A2) (SEQ ID NO: 68) or HRSV(Memphis-37) (SEQ ID NO: 8937 or 11726). Moreover, the abbreviations for suitable RSV F protein antigen designs in Table 1 are used throughout the description of the invention (e.g., “F0”, “F-del”, “F0_DSCav1”, “F-del_DSCav1”, “F_DSCav1_mut1”, “F-del_DSCav1_mut1”, “F_DSCav1_mut2”, “F-del_DSCav1_mut2”, “F_DSCav1_mut3”, “F-del_DSCav1_mut3”, “F_DSCav1_mut4”, “F-del_DSCav1_mut4”, “F_DSCav1_mut5”, “F-del_DSCav1_mut5”, “F_DSCav1_mut6”, “F-del_DSCav1_mut6”, “F_DSCav1_mut7”, “F-del_DSCav1_mut7 ”, “F_DSCav1_mut8”, “F-del_DSCav1_mut8”, “F_DSCav1_mut0”, “F-del_DSCav1_mut0”). Column A of Table 1 provides protein SEQ ID NOs of respective RSV F protein antigen designs derived from HRSV(A2); Column B of Table 1 provides protein SEQ ID NOs of respective RSV F protein antigen designs derived from HRSV(Memphis-37). Notably, the description of the invention explicitly includes the information provided under <223> identifier of the ST25 sequence listing of the present application.

TABLE 1 Preferred RSV F protein antigen designs Antigen Name Protein design description A B F0 aa1-574, 68 8937, full-length RSV F0 precursor 11726 F-del aa1-553, 483 9653, deletion of aa 554-574 of the C-terminus 12095 F0_DSCav1 aa1-574, 898 12464 aa substitutions: S155C, S290C, S190F, and V207L F-del_DSCav1 aa1-553, 1267 12833 deletion of aa 554-574 of the C-terminus, aa substitutions: S155C, S290C, S190F, and V207L F_DSCav1_mut0 aa1-103 - GS(linker) - aa145-574 3850 13940 F2-linker-F1 construct aa substitutions: S155C, S290C, S190F, and V207L F-del_DSCav1_mut0 aa1-103 - GS(linker) - aa145-553 4219 14309 deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct aa substitutions: S155C, S290C, S190F, and V207L F_DSCav1_mut1 aa1-103 - GS(linker) - aa145-574, 1636 14678 F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; S46G, A149C, S215P, Y458C, K465Q F-del_DSCav1_mut1 aa1-103 - GS(linker) - aa145-553 2005 15047 deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; S46G, A149C, S215P, Y458C, K465Q F_DSCav1_mut2 aa1-103 - GS(linker) - aa145-574 2374 15416 F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; S46G, E92D, A149C, S215P, Y458C, K465Q F-del_DSCav1_mut2 aa1-103 - GS(linker) - aa145-553 2743 15785 deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; S46G, E92D, A149C, S215P, Y458C, K465Q F_DSCav1_mut3 aa1-103 - GS(linker) - aa145-574 3112 13202 F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; S46G, N67I, E92D, A149C, S215P, Y458C, K465Q F-del_DSCav1_mut3 aa1-103 - GS(linker) - aa145-553 3481 13571 deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; S46G, N67I, E92D, A149C, S215P, Y458C, K465Q F_DSCav1_mut4 aa1-103 - GS(linker) - aa145-574 4588 16154 F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; A149C, Y458C F-del_DSCav1_mut4 aa1-103 - GS(linker) - aa145-553 4957 16523 deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; A149C, Y458C F_DSCav1_mut5 aa1-103 - GS(linker) - aa145-574 5326 16892 F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; N183GC, N428C F-del_DSCav1_mut5 aa1-103 - GS(linker) - aa145-553 5695 17261 deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; N183GC, N428C F_DSCav1_mut6 aa1-103 - GS(linker) - aa145-574 6064 17630 F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; Q98C, Q361C, S46G, E92D, L95M, S215P, I217P, I221M, R429K, K465Q F-del_DSCav1_mut6 aa1-103 - GS(linker) - aa145-553 6433 17999 deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; Q98C, Q361C, S46G, E92D, L95M, S215P, I217P, I221M, R429K, K465Q F_DSCav1_mut7 aa1-103 - GS(linker) - aa145-574 6802 18368 F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; Q98C, Q361C, L95M, I221M, R429K F-del_DSCav1_mut7 aa1-103 - GS(linker) - aa145-553 7171 18737 deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; Q98C, Q361C, L95M, I221M, R429K F_DSCav1_mut8 aa1-103 - GS(linker) - aa145-574 7540 19106 F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; N183GC, N428C, S46G, N67I, E92D, S215P, K465Q F-del_DSCav1_mut8 aa1-103 - GS(linker) - aa145-553 7909 19475 deletion of aa 554-574 of the C-terminus, F2-linker-F1 construct, aa substitutions: S155C, S290C, S190F, and V207L; N183GC, N428C, S46G, N67I, E92D, S215P, K465Q

In particularly preferred embodiments, the artificial RNA according to the first aspect encodes at least one antigenic peptide or protein derived from a RSV fusion (F) protein, wherein the RSV F protein is selected from F0, F-del, F0_DSCav1, F_DSCav1_mut0, F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3, F_DSCav1_mut4, F_DSCav1_mut5, F_DSCav1_mut6, F_DSCav1_mut7, F_DSCav1_mut8, F-del_DSCav1, F-del_DSCav1_mut0, F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut4, F-del_DSCav1_mut5, F-del_DSCav1_mut6, F-del_DSCav1_mut7, F-del_DSCav1_mut8, or a fragment or a variant thereof (see e.g. Table 1).

Particularly preferred and advantageous in the context of the invention are RSV F proteins selected from F-del_DSCav1, F-del_DSCav1_mut0, F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut4, F-del_DSCav1_mut5, F-del_DSCav1_mut6, F-del_DSCav1_mut7, F-del_DSCav1_mut8 or a fragment or a variant thereof (see e.g. Table 1).

In preferred embodiments, the artificial RNA of the first aspect comprises at least one coding sequence encoding at least one antigenic peptide or protein comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 19475 (see e.g. Table 1), or 8279-9683 or a fragment or variant of any of these sequences. Additional information regarding each of these suitable amino acid sequences encoding RSV proteins may also be derived from the or 99% identical to any one of SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106, sequence listing, in particular from the details provided therein under identifier <223> as explained in the following.

In preferred embodiments, the artificial RNA of the first aspect comprises at least one coding sequence encoding at least one antigenic peptide or protein comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106, 19475 (see e.g. Table 1) or a fragment or variant of any of these sequences. Additional information regarding each of these suitable amino acid sequences encoding RSV proteins may also be derived from the sequence listing, in particular from the details provided therein under identifier <223> as explained in the following.

In particularly preferred embodiments, the artificial RNA of the first aspect comprises at least one coding sequence encoding at least one antigenic peptide or protein comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 1267, 2005, 2743, 3481, 4219, 4957, 5695, 6433, 7171, 7909, 12833, 14309, 15047, 15785, 13571, 16523, 17261, 17999, 18737, 19475 (see e.g. Table 1) or a fragment or variant of any of these sequences. Additional information regarding each of these suitable amino acid sequences encoding RSV proteins may also be derived from the sequence listing, in particular from the details provided therein under identifier <223> as explained in the following.

In other embodiments, the artificial RNA according to the first aspect comprises at least one coding sequence encoding at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 8279-9683 a fragment or variant of any of these sequences. Additional information regarding each of these suitable amino acid sequences encoding RSV proteins may also be derived from the sequence listing, in particular from the details provided therein under identifier <223> as explained in the following.

In other embodiments, the artificial RNA as defined herein comprises at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-1428 of patent application WO2014/160463 or a fragment or variant of any of these sequences. In this context, SEQ ID NOs: 1-1428 of patent application WO2014/160463 and the disclosure related thereto are herewith incorporated by reference.

In other embodiments, the artificial RNA as defined herein comprises at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-11 of patent application WO2015/024668 or a fragment or variant of any of these sequences. In this context, SEQ ID NOs: 1-11 of patent application WO2015/024668 and the disclosure related thereto are herewith incorporated by reference.

In other embodiments, the artificial RNA as defined herein comprises at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 243, or 245 of patent application WO2017/070622 or a fragment or variant of any of these sequences. In this context SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 243, or 245, of patent application WO2017/070622 and the disclosure related thereto are herewith incorporated by reference.

In other embodiments, the artificial RNA as defined herein comprises at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV comprising or consisting of at least one amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-65, 81-95, 110-116 of patent application WO2017/172890 or a fragment or variant of any of these sequences. In this context SEQ ID NOs: 1-65, 81-95, 110-116, of patent application WO2017/172890 and the disclosure related thereto are herewith incorporated by reference.

According to another preferred embodiment, the artificial RNA of the invention encodes at least one antigenic peptide or protein as defined above and may additionally encode at least one further heterologous peptide or protein element.

Suitably, the at least one further peptide or protein element may promote secretion of the encoded antigenic peptide or protein of the invention (e.g. via secretory signal sequences), promote anchoring of the encoded antigenic peptide or protein of the invention in the plasma membrane (e.g. via transmembrane elements), promote formation of antigen complexes (e.g. via multimerization domains), promote virus-like particle formation (VLP forming sequence). In addition, the artificial nucleic acid sequence according to the present invention may additionally encode peptide linker elements, self-cleaving peptides, immunologic adjuvant sequences or dendritic cell targeting sequences. Suitable multimerization domains may be selected from the list of amino acid sequences according to SEQ ID NOs: 1116-1167 of the patent application WO2017/081082, or fragments or variants of these sequences. Trimerization and tetramerization elements may be selected from e.g. engineered leucine zippers (engineered α-helical coiled coil peptide that adopt a parallel trimeric state), fibritin foldon domain from enterobacteria phage T4, GCN4pII, GCN4-pLI, and p53. In that context, fibritin foldon domain from enterobacteria phage T4, GCN4pII, GCN4-pLI, and p53 are preferred. Suitable transmembrane elements may be selected from the list of amino acid sequences according to SEQ ID NOs: 1228-1343 of the patent application WO2017/081082, or fragments or variants of these sequences. Suitable VLP forming sequences may be selected from the list of amino acid sequences according to SEQ ID NOs: 1168-1227 of the patent application WO2017/081082, or fragments or variants of these sequences. Suitable peptide linkers may be selected from the list of amino acid sequences according to SEQ ID NOs: 1509-1565 of the patent application WO2017/081082, or fragments or variants of these sequences. Suitable self-cleaving peptides may be selected from the list of amino acid sequences according to SEQ ID NOs: 1434-1508 of the patent application WO2017/081082, or fragments or variants of these sequences. Suitable immunologic adjuvant sequences may be selected from the list of amino acid sequences according to SEQ ID NOs: 1360-1421 of the patent application WO2017/081082, or fragments or variants of these sequences. Suitable dendritic cell (DCs) targeting sequences may be selected from the list of amino acid sequences according to SEQ ID NOs: 1344-1359 of the patent application WO2017/081082, or fragments or variants of these sequences. Suitable secretory signal peptides may be selected from the list of amino acid sequences according to SEQ ID NOs: 1-1115 and SEQ ID NO: 1728 of the patent application WO2017/081082, or fragments or variants of these sequences. On nucleic acid level, any nucleic acid sequence (e.g. RNA sequence) may be selected which encodes such amino acid sequences. In this context, the disclosure of WO2017/081082 is herewith incorporated by reference. The heterologous secretory signal sequence may increase the secretion of the encoded antigenic peptide or protein.

According to embodiments, the secretory signal sequence comprises an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 21329-21362 or a fragment or variant of any of these sequences. Additional information regarding each of these suitable amino acid sequences encoding secretory signal sequences may also be derived from the sequence listing, in particular from the details provided therein under identifier <223>.

According to preferred embodiments, the artificial nucleic acid, particularly the artificial RNA comprises at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV as defined herein, preferably derived from RSV F protein, or fragments and variants thereof. In that context, any coding sequence encoding at least one antigenic peptide or protein derived from RSV, preferably derived from RSV F protein, or fragments and variants thereof may be understood as suitable coding sequence and may therefore be comprised in the artificial RNA of the first aspect.

In preferred embodiments, the artificial RNA of the first aspect may comprise or consist of at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV F protein as defined herein, preferably encoding any one of SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106, 19475 or fragments of variants thereof. It has to be understood that, on nucleic acid level, any nucleic acid sequence, in particular, any RNA sequence which encodes an amino acid sequences being identical to SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106, 19475 or fragments or variants thereof, or any nucleic acid sequence (e.g. DNA sequence, RNA sequence) which encodes amino acid sequences being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106, 19475 or fragments or variants thereof, may be selected and may accordingly be understood as suitable coding sequence and may therefore be comprised in the artificial RNA of the first aspect of the invention.

In other embodiments, the artificial RNA of the first aspect may comprise or consist of at least one coding sequence encoding any one of SEQ ID NOs: 8279-9683 or fragments of variants thereof. It has to be understood that, on nucleic acid level, any nucleic acid sequence, in particular, any RNA sequence which encodes an amino acid sequences being identical to SEQ ID NOs: 8279-9683 or fragments or variants thereof, or any nucleic acid sequence (e.g. DNA sequence, RNA sequence) which encodes amino acid sequences being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 8279-9683 or fragments or variants thereof, may be selected and may accordingly be understood as suitable coding sequence and may therefore be comprised in the artificial RNA of the first aspect of the invention.

In other embodiments, the artificial RNA of the first aspect may comprise or consist of at least one coding sequence encoding any one of SEQ ID NOs: 1-1428 of patent application WO2014/160463 or fragments of variants thereof. It has to be understood that, on nucleic acid level, any nucleic acid sequence, in particular, any RNA sequence which encodes an amino acid sequences being identical to SEQ ID NOs: 1-1428 of patent application WO2014/160463 or fragments or variants thereof, or any nucleic acid sequence (e.g. DNA sequence, RNA sequence) which encodes amino acid sequences being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-1428 of patent application WO2014/160463 or fragments or variants thereof, may be selected and may accordingly be understood as suitable coding sequence and may therefore be comprised in the artificial RNA of the first aspect of the invention.

In other embodiments, the artificial RNA of the first aspect may comprise or consist of at least one coding sequence encoding any one of SEQ ID NOs: 1-11 of patent application WO2015/024668 or fragments of variants thereof. It has to be understood that, on nucleic acid level, any nucleic acid sequence, in particular, any RNA sequence which encodes an amino acid sequences being identical to SEQ ID NOs: 1-11 of patent application WO2015/024668 or fragments or variants thereof, or any nucleic acid sequence (e.g. DNA sequence, RNA sequence) which encodes amino acid sequences being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-11 of patent application WO2015/024668 or fragments or variants thereof, may be selected and may accordingly be understood as suitable coding sequence and may therefore be comprised in the artificial RNA of the first aspect of the invention.

In other embodiments, the artificial RNA of the first aspect may comprise or consist of at least one coding sequence encoding any one of SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 243, or 245 of patent application WO2017/070622 or fragments of variants thereof. It has to be understood that, on nucleic acid level, any nucleic acid sequence, in particular, any RNA sequence which encodes an amino acid sequences being identical to SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 243, or 245 of patent application WO2017/070622 or fragments or variants thereof, or any nucleic acid sequence (e.g. DNA sequence, RNA sequence) which encodes amino acid sequences being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 243, or 245 of patent application WO2017/070622 or fragments or variants thereof, may be selected and may accordingly be understood as suitable coding sequence and may therefore be comprised in the artificial RNA of the first aspect of the invention.

In other embodiments, the artificial RNA of the first aspect may comprise or consist of at least one coding sequence encoding any one of SEQ ID NOs: 1-65, 81-95, 110-116 of patent application WO2017/172890 or fragments of variants thereof. It has to be understood that, on nucleic acid level, any nucleic acid sequence, in particular, any RNA sequence which encodes an amino acid sequences being identical to SEQ ID NOs: 1-65, 81-95, 110-116 of patent application WO2017/172890 or fragments or variants thereof, or any nucleic acid sequence (e.g. DNA sequence, RNA sequence) which encodes amino acid sequences being at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-65, 81-95, 110-116 of patent application WO2017/172890 or fragments or variants thereof, may be selected and may accordingly be understood as suitable coding sequence and may therefore be comprised in the artificial RNA of the first aspect of the invention.

Suitably, in particularly preferred embodiments, the artificial RNA of the first aspect comprises a coding sequence located between said 5′-UTR and said 3′-UTR, preferably downstream of said 5′-UTR and upstream of said 3′-UTR.

In preferred embodiments, the artificial RNA of the first aspect comprises a coding sequence that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 69-482, 484-897, 899-1266, 1268-1635, 1637-2004, 2006-2373, 2375-2742, 2744-3111, 3113-3480, 3482-3849, 3851-4218, 4220-4587, 4589-4956, 4958-5325, 5327-5694, 5696-6063, 6065-6432, 6434-6801, 6803-7170, 7172-7539, 7541-7908, 7910-8277, 8278, 11727-12094, 12096-12463, 12465-12832, 12834-13201, 13941-14308, 14310-14677, 14679-15046, 15048-15415, 15417-15784, 15786-16153, 13203-13570, 13572-13939, 16155-16522, 16524-16891, 16893-17260, 17262-17629, 17631-17998, 18000-17998, 18369-18736, 18738-19105, 19107-19474, 19476-19843, 21363-21706 or a fragment or a fragment or variant of any of these sequences (see also Table 3-6). Additional information regarding each of these suitable nucleic acid sequences encoding may also be derived from the sequence listing, in particular from the details provided therein under identifier <223>.

In particularly preferred embodiments, the artificial RNA of the first aspect comprises a coding sequence that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 69-77, 484-492, 899-906, 1268-1275, 1637-1644, 2006-2013, 2375-2382, 2744-2751, 3113-3120, 3482-3489, 3851-3858, 4220-4227, 4589-4596, 4958-4965, 5327-5334, 5696-5703, 6065-6072, 6434-6441, 6803-6810, 7172-7179, 7541-7548, 7910-7917, 21363-21384, 11727-11734, 12096-12103, 12465-12472, 12834-12841, 13941-13948, 14310-14317, 14679-14686, 15048-15055, 15417-15424, 15786-15793, 13203-13210, 13572-13579, 16155-16162, 16524-16531, 16893-16900, 17262-17269, 17631-17638, 18000-18007, 18369-18376, 18738-18745, 19107-19114, 19476-19483, 21389-21410 or a fragment or a fragment or variant of any of these sequences (see also Table 3 and 4). Additional information regarding each of these suitable nucleic acid sequences encoding may also be derived from the sequence listing, in particular from the details provided therein under identifier <223>.

In other embodiments, the artificial RNA of the first aspect comprises a coding sequence that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 383-388 of patent application WO2014/160463 or a fragment or a fragment or variant of any of these sequences.

In other embodiments, the artificial RNA of the first aspect comprises a coding sequence that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 12-22 of patent application WO2015/024668 or a fragment or a fragment or variant of any of these sequences.

In other embodiments, the artificial RNA of the first aspect comprises a coding sequence that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1, 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 242, 244, 246, 257, 258-280 of patent application WO2017/070622 or a fragment or a fragment or variant of any of these sequences.

In other embodiments, the artificial RNA of the first aspect comprises a coding sequence that comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 96-99 of patent application WO2017/172890 or a fragment or a fragment or variant of any of these sequences.

According to preferred embodiments, the artificial RNA is a modified and/or stabilized artificial RNA.

According to preferred embodiments, the artificial RNA of the present invention may thus be provided as a “stabilized artificial RNA” that is to say an RNA showing improved resistance to in vivo degradation and/or an artificial RNA showing improved stability in vivo, and/or an artificial RNA showing improved translatability in vivo. In the following, specific suitable modifications in this context are described which are suitably to “stabilize” the artificial RNA.

Such stabilization may be effected by providing a “dried RNA” and/or a “purified RNA” as specified herein. Alternatively or in addition to that, such stabilization can be effected, for example, by a modified phosphate backbone of the artificial RNA of the present invention. A backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in the RNA are chemically modified. Nucleotides that may be preferably used in this connection contain e.g. a phosphorothioate-modified phosphate backbone, preferably at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom. Stabilized RNAs may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form. Such backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5-O-(1-thiophosphate)).

In the following, suitable modifications are described that are capable of “stabilizing” the artificial RNA of the invention.

According to embodiments, the artificial RNA according to the invention is a modified artificial RNA, wherein the modification refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications.

In this context, a modified artificial RNA as defined herein may contain nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications or base modifications. A backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides contained in a nucleic acid, e.g. an artificial RNA, are chemically modified. A sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the RNA as defined herein. Furthermore, a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides of the RNA. In this context, nucleotide analogues or modifications are preferably selected from nucleotide analogues which are applicable for transcription and/or translation.

In particularly preferred embodiments of the present invention, the nucleotide analogues/modifications which may be incorporated into a modified nucleic acid or particularly into a modified RNA as described herein are preferably selected from 2-amino-6-chloropurineriboside-5′-triphosphate, 2-Aminopurine-riboside-5′-triphosphate; 2-aminoadenosine-5′-triphosphate, 2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate, 2′-O-Methyl-inosine-5′-triphosphate 4-thiouridine-5′-triphosphate, 5-aminoallylcytidine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, 5-bromouridine-5′-triphosphate, 5-Bromo-2′-deoxycytidine-5′-triphosphate, 5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 5-Iodo-2′-deoxyuridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate, 5-Propynyl-2′-deoxycytidine-5′-triphosphate, 5-Propynyl-2′-deoxyuridine-5′-triphosphate, 6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate, 7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, xanthosine-5′-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine, 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, 5′-O-(1-thiophosphate)-pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine. Particularly preferred and suitable in the context of the invention are pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine. Accordingly, the artificial RNA as defined herein may comprise at least one modified nucleotide selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine.

In preferred embodiments, the artificial RNA of the invention comprises at least one coding sequence, wherein the at least one coding sequence is a pseudouridine (ψ) modified coding sequence.

Accordingly, in preferred embodiments, the artificial RNA of the invention, or the at least one coding sequence, comprises a nucleic acid sequence wherein at least one or more than one, preferably wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides

In further preferred embodiments, the artificial RNA of the invention comprises at least one coding sequence, wherein the at least one coding sequence is a N1-methylpseudouridine (m1ψ) modified coding sequence.

Accordingly, in preferred embodiments, the artificial RNA of the invention, or the at least one coding sequence, comprises a nucleic acid sequence wherein at least one or more than one, preferably wherein all uracil nucleotides are replaced by N1-methylpseudouridine (m1ψ) nucleotides

In preferred embodiments, the artificial RNA of the invention comprises at least one coding sequence, wherein the at least one coding sequence is a codon modified coding sequence.

In preferred embodiments, the at least one coding sequence of the invention is a codon modified coding sequence, wherein the amino acid sequence encoded by the at least one codon modified coding sequence is preferably not being modified compared to the amino acid sequence encoded by the corresponding wild type coding sequence.

The term “codon modified coding sequence” relates to coding sequences that differ in at least one codon (triplets of nucleotides coding for one amino acid) compared to the corresponding wild type coding sequence. Suitably, a codon modified coding sequence in the context of the invention may show improved resistance to in vivo degradation and/or improved stability in vivo, and/or improved translatability in vivo. Codon modifications in the broadest sense make use of the degeneracy of the genetic code wherein multiple codons may encode the same amino acid and may be used interchangeably (cf. Table 2) to optimize/modify the coding sequence for in vivo applications as outlined above.

In particularly preferred embodiments of the first aspect, the at least one sequence is a codon modified coding sequence, wherein the codon modified coding sequence is selected from C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified coding sequence, and G/C optimized coding sequence, or any combination thereof, or any combination thereof.

According to preferred embodiments, the artificial RNA of the invention may be modified, wherein the C content of the at least one coding sequence may be increased, preferably maximized, compared to the C content of the corresponding wild type coding sequence (herein referred to as “C maximized coding sequence”). The amino acid sequence encoded by the C maximized coding sequence of the RNA is preferably not modified as compared to the amino acid sequence encoded by the respective wild type nucleic acid coding sequence. The generation of a C maximized nucleic acid sequences may suitably be carried out using a modification method according to WO2015/062738. In this context, the disclosure of WO2015/062738 is included herewith by reference. Throughout the disclosure of the invention, including the <223> identifier of the sequence listing, C maximized coding sequences of suitable RSV nucleic acid sequences are indicated by the abbreviation “opt2”.

According to embodiments, the artificial RNA of the present invention may be modified, wherein the G/C content of the at least one coding sequence of the invention may be modified compared to the G/C content of the corresponding wild type coding sequence (herein referred to as “G/C content modified coding sequence”). In this context, the terms “G/C optimization” or “G/C content modification” relate to a nucleic acid, preferably an artificial nucleic acid of the invention that comprises a modified, preferably an increased number of guanosine and/or cytosine nucleotides as compared to the corresponding wild type nucleic acid sequence. Such an increased number may be generated by substitution of codons containing adenosine or thymidine nucleotides by codons containing guanosine or cytosine nucleotides. If the enriched G/C content occurs in a coding sequence of DNA or RNA, it makes use of the degeneracy of the genetic code. In particular, in case of RNA, sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content. The amino acid sequence encoded by the G/C content modified coding sequence of the nucleic acid sequence is preferably not modified as compared to the amino acid sequence encoded by the respective wild type nucleic acid coding sequence. Preferably, the G/C content of the coding sequence of the artificial nucleic acid sequence, e.g. the RNA sequence of the present invention is increased by at least 10%, preferably by at least 20%, more preferably by at least 30%, most preferably by at least 40% compared to the G/C content of the coding sequence of the corresponding wild type nucleic acid sequence (e.g. RNA sequence), which codes for a RSV antigen as defined herein or a fragment or variant thereof.

According to preferred embodiments, the artificial RNA of the present invention may be modified, wherein the G/C content of the at least one coding sequence of the invention may be optimized compared to the G/C content of the corresponding wild type coding sequence (herein referred to as “G/C content optimized coding sequence”). “Optimized” in that context refers to a coding sequence wherein the G/C content is preferably increased to the essentially highest possible G/C content. The amino acid sequence encoded by the G/C content optimized coding sequence of the nucleic acid sequence is preferably not modified as compared to the amino acid sequence encoded by the respective wild type nucleic acid coding sequence. The generation of a G/C content optimized nucleic RNA sequence may suitably be carried out using a G/C content optimization method according to WO2002/098443. In this context, the disclosure of WO2002/098443 is included in its full scope in the present invention. Throughout the disclosure of the invention, including the <223> identifier of the sequence listing, G/C optimized coding sequences of suitable RSV nucleic acid sequences are indicated by the abbreviation “opt1, opt5, opt6, opt11”.

According to embodiments, the artificial RNA of the invention may be modified, wherein the codons in the at least one coding sequence of the invention may be adapted to human codon usage (herein referred to as “human codon usage adapted coding sequence”). Codons encoding the same amino acid occur at different frequencies in a subject, e.g. a human. Accordingly, the coding sequence of the artificial RNA is preferably modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage e.g. as shown in Table 2. For example, in the case of the amino acid Ala, the wild type coding sequence is preferably adapted in a way that the codon “GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with a frequency of 0.10 etc. (see Table 2). Accordingly, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of the artificial nucleic acid of the invention to obtain sequences adapted to human codon usage. Throughout the disclosure of the invention, including the <223> identifier of the sequence listing, human codon usage adapted coding sequences of suitable RSV nucleic acid sequences are indicated by the abbreviation “opt3”.

TABLE 2 Human codon usage table with frequencies indicated for each amino acid Amino acid codon frequency Ala GCG 0.10 Ala GCA 0.22 Ala GCT 0.28 Ala GCC* 0.40 Cys TGT 0.42 Cys TGC* 0.58 Asp GAT 0.44 Asp GAC* 0.56 Glu GAG* 0.59 Glu GAA 0.41 Phe TTT 0.43 Phe TTC* 0.57 Gly GGG 0.23 Gly GGA 0.26 Gly GGT 0.18 Gly GGC* 0.33 His CAT 0.41 His CAC* 0.59 Ile ATA 0.14 Ile ATT 0.35 Ile ATC* 0.52 Lys AAG* 0.60 Lys AAA 0.40 Leu TTG 0.12 Leu TTA 0.06 Leu CTG* 0.43 Leu CTA 0.07 Leu CTT 0.12 Leu CTC 0.20 Met ATG* 1 Asn AAT 0.44 Asn AAC* 0.56 Pro CCG 0.11 Pro CCA 0.27 Pro CCT 0.29 Pro CCC* 0.33 Gln CAG* 0.73 Gln CAA 0.27 Arg AGG 0.22 Arg AGA* 0.21 Arg CGG 0.19 Arg CGA 0.10 Arg CGT 0.09 Arg CGC 0.19 Ser AGT 0.14 Ser AGC* 0.25 Ser TCG 0.06 Ser TCA 0.15 Ser TCT 0.18 Ser TCC 0.23 Thr ACG 0.12 Thr ACA 0.27 Thr ACT 0.23 Thr ACC* 0.38 Val GTG* 0.48 Val GTA 0.10 Val GTT 0.17 Val GTC 0.25 Trp TGG* 1 Tyr TAT 0.42 Tyr TAC* 0.58 Stop TGA* 0.61 Stop TAG 0.17 Stop TAA 0.22 *most frequent human codon

According to embodiments, the artificial RNA of the present invention may be modified, wherein the codon adaptation index (CAI) may be increased or preferably maximised in the at least one coding sequence of the invention (herein referred to as “CAI maximized coding sequence”). Accordingly, it is preferred that all codons of the wild type nucleic acid sequence that are relatively rare in e.g. a human cell are exchanged fora respective codon that is frequent in the e.g. a human cell, wherein the frequent codon encodes the same amino acid as the relatively rare codon. Suitably, the most frequent codons are used for each encoded amino acid (see Table 2, most frequent human codons are marked with asterisks). Suitably, the artificial RNA of the invention comprises at least one coding sequence, wherein the codon adaptation index (CAI) of the at least one coding sequence is at least 0.5, at least 0.8, at least 0.9 or at least 0.95. Most preferably, the codon adaptation index (CAI) of the at least one coding sequence is 1. For example, in the case of the amino acid Ala, the wild type coding sequence is adapted in a way that the most frequent human codon “GCC” is always used for said amino acid. Accordingly, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of the artificial RNA of the invention to obtain CAI maximized coding sequences. Throughout the disclosure of the invention including the <223> identifier of the sequence listing, CAI maximized coding sequences of suitable RSV nucleic acid sequences are indicated by the abbreviation “opt4”.

Accordingly, in a particularly preferred embodiment, the artificial RNA of the first aspect comprises at least one coding sequence comprising a codon modified nucleic acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a codon modified nucleic acid sequence selected from the group consisting of SEQ ID NOs: 70-77, 485-492, 899-906, 1268-1275, 1637-1644, 2006-2013, 2375-2382, 2744-2751, 3113-3120, 3482-3489, 3851-3858, 4220-4227, 4589-4596, 4958-4965, 5327-5334, 5696-5703, 6065-6072, 6434-6441, 6803-6810, 7172-7179, 7541-7548, 7910-7917, 11728-11734, 12097-12103, 12465-12472, 12834-12841, 13941-13948, 14310-14317, 14679-14686, 15048-15055, 15417-15424, 15786-15793, 13203-13210, 13572-13579, 16155-16162, 16524-16531, 16893-16900, 17262-17269, 17631-17638, 18000-18007, 18369-18376, 18738-18745, 19107-19114, 19476-19483, 21363-21384, 21389-21410 or a fragment or variant of any of these sequences (see also Table 3 and 4). Additional information regarding each of these suitable nucleic acid sequences encoding may also be derived from the sequence listing, in particular from the details provided therein under identifier <223>.

In particularly preferred embodiment, the artificial RNA of the first aspect comprises at least one coding sequence comprising a codon modified nucleic acid sequence which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the G/C optimized or G/C content modified nucleic acid sequence according to the SEQ ID NOs: 70-71, 75-77, 485-486, 490-492, 899-900, 904-906, 1268-1269, 1273-1275, 1637-1638, 1642-1644, 2006-2007, 2011-2013, 2375-2376, 2380-2382, 2744-2745, 2749-2751, 3113-3114, 3118-3120, 3482-3483, 3487-3489, 3852, 4221, 4590, 4959, 5328, 5697, 6066, 6435, 6804, 7173, 7542, 7911, 3856-3858, 4225-4227, 4594-4596, 4963-4965, 5332-5334, 5701-5703, 6070-6072, 6439-6441, 6808-6810, 7177-7179, 7546-7548, 7915-7917, 11728, 11732-11734, 12097, 12101-12103, 12465, 12466, 12470-12472, 12834, 12835, 12839-12841, 13941, 13942, 13946-13948, 14310, 14311, 14315-14317, 14679, 14680, 14684-14686, 15048, 15049, 15053-15055, 15417, 15418, 15422-15424, 15786, 15787, 15791-15793, 13203, 13204, 13208-13210, 13572, 13573, 13577-13579, 16155, 16156, 16160-16162, 16524, 16525, 16529-16531, 16893, 16894, 16898-16900, 17262, 17263, 17267-17269, 17631, 17632, 17636-17638, 18000, 18001, 18005-18007, 18369, 18370, 18374-18376, 18738, 18739, 18743-18745, 19107, 19108, 19112-19114, 19476, 19477, 19481-19483, 21363-21384, 21389-21410 or a fragment or variant of any of these sequences (see also Table 3 and 4; opt1, 5, 6, 11). Additional information regarding each of these suitable nucleic acid sequences encoding may also be derived from the sequence listing, in particular from the details provided therein under identifier <223>.

In preferred embodiment, the artificial RNA of the invention comprises at least one coding sequence comprising a codon modified nucleic acid sequence which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the human codon usage adapted nucleic acid sequence according to the SEQ ID NOs: 73, 488, 902, 1271, 1640, 2009, 2378, 2747, 3116, 3485, 3854, 4223, 4592, 4961, 5330, 5699, 6068, 6437, 6806, 7175, 7544, 7913, 11730, 12099, 12468, 12837, 13944, 14313, 14682, 15051, 15420, 15789, 13206, 13575, 16158, 16527, 16896, 17265, 17634, 18003, 18372, 18741, 19110, 19479 or a fragment or variant of any of these sequences (see also Table 3 and 4; opt3). Additional information regarding each of these suitable nucleic acid sequences encoding may also be derived from the sequence listing, in particular from the details provided therein under identifier <223>.

In particularly preferred embodiment, the artificial RNA of the first aspect comprises at least one coding sequence comprising a codon modified nucleic acid sequence which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the C maximized nucleic acid sequence according to the SEQ ID NOs: 72, 487, 901, 1270, 1639, 2008, 2377, 2746, 3115, 3484, 3853, 4222, 4591, 4960, 5329, 5698, 6067, 6436, 6805, 7174, 7543, 7912, 11729, 12098, 12467, 12836, 13943, 14312, 14681, 15050, 15419, 15788, 13205, 13574, 16157, 16526, 16895, 17264, 17633, 18002, 18371, 18740, 19109, 19478 or a fragment or variant of any of these sequences (see also Table 3 and 4; opt2).

Additional information regarding each of these suitable nucleic acid sequences encoding may also be derived from the sequence listing, in particular from the details provided therein under identifier <223>.

In preferred embodiment, the artificial RNA of the invention comprises at least one coding sequence comprising a codon modified nucleic acid sequence which is identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the CAI maximized nucleic acid sequence according to the SEQ ID NOs 74, 489, 903, 1272, 1641, 2010, 2379, 2748, 3117, 3486, 3855, 4224, 4593, 4962, 5331, 5700, 6069, 6438, 6807, 7176, 7545, 7914, 11731, 12100, 12469, 12838, 13945, 14314, 14683, 15052, 15421, 15790, 13207, 13576, 16159, 16528, 16897, 17266, 17635, 18004, 18373, 18742, 19111, 19480 or a fragment or variant of any of these sequences (see also Table 3 and 4; opt4). Additional information regarding each of these suitable nucleic acid sequences encoding may also be derived from the sequence listing, in particular from the details provided therein under identifier <223>.

In embodiments, the A/U content in the environment of the ribosome binding site of the artificial nucleic acid, particularly the artificial RNA of the invention may be increased compared to the A/U content in the environment of the ribosome binding site of its respective wild type nucleic acid. This modification (an increased A/U content around the ribosome binding site) increases the efficiency of ribosome binding to the nucleic acid, preferably the RNA. An effective binding of the ribosomes to the ribosome binding site in turn has the effect of an efficient translation of the RNA. Accordingly, in a particularly preferred embodiment, the artificial nucleic acid of the invention comprises a ribosome binding site, also referred to as “Kozak sequence” identical to or at least 80%, 85%, 90%, 95% identical to any one of the sequences SEQ ID NOs: 41, 42, or fragments or variants thereof.

Preferred RSV polypeptide and nucleic acid coding sequences (“cds”) are provided in Table 3A and 3B and Table 4A and 4B.

In Table 3A and 3B, Columns A to J represent specific suitable constructs of the invention derived from RSV Fusion (F) protein, wherein Column A provides suitable sequences for F0, Column B provides suitable sequences for F-del, Column C provides suitable sequences for F0_DSCav1, Column D provides suitable sequences for F-del_DSCav1, Column E provides suitable sequences for F_DSCav1_mut1, Column F provides suitable sequences for F-del_DSCav1_mut1, Column G provides suitable sequences for F_DSCav1_mut2, Column H provides suitable sequences for F-del_DSCav1_mut2, Column I provides suitable sequences for F_DSCav1_mut3, Column J provides suitable sequences for F-del_DSCav1_mut3. The specific protein SEQ ID NOs as provided in the sequence listing are in row 2 (“PRT”). The SEQ ID NOs of corresponding wild type/non-modified coding sequences are provided in row 3 (“wt”). The SEQ ID NOs of corresponding codon modified coding sequences for each protein construct are provided in row 4 to row 10 (“opt1”, “opt2”, “opt3”, “opt4”, “opt5”, “opt6”, “opt11”). In Table 3A, coding sequences derived from HRSV(A2) are provided, in Table 3B coding sequences derived from HRSV(Memphis-37) are provided. Further information is provided in the <223> identifier for each of the respective SEQ ID NO in the sequence listing.

TABLE 3A Preferred coding sequences encoding RSV F (columns A-J), derived from HRSV(A2) A B C D E F G H I J PRT 68 483 898 1267 1636 2005 2374 2743 3112 3481 wt 69 484 opt1 70, 71, 485, 899, 1268, 1637, 2006, 2375, 2744, 3113, 3482, 21363 486, 900, 1269, 1638, 2007, 2376, 2745, 3114, 3483, 21364 21365 21366 21369 21370 21371 21372 21373 21374 opt2 72 487 901 1270 1639 2008 2377 2746 3115 3484 opt3 73 488 902 1271 1640 2009 2378 2747 3116 3485 opt4 74 489 903 1272 1641 2010 2379 2748 3117 3486 opt5 75 490 904 1273 1642 2011 2380 2749 3118 3487 opt6 76 491 905 1274 1643 2012 2381 2750 3119 3488 opt11 77 492 906 1275 1644 2013 2382 2751 3120 3489

TABLE 3B Preferred coding sequences encoding RSV F (columns A-J), derived from HRSV(Memphis-37) A B C D E F G H I J PRT 11726 12095 12464 12833 13940 14309 14678 15047 15416 15785 wt 11727 12096 opt1 11728, 12097, 12465, 12834, 13941, 14310, 14679, 15048, 15417, 15786, 21389 21390 12466, 12835, 13942, 14311, 14680, 15049, 15418, 15787, 21391 21392 21395 21396 21397 21398 21399 21400 opt2 11729 12098 12467 12836 13943 14312 14681 15050 15419 15788 opt3 11730 12099 12468 12837 13944 14313 14682 15051 15420 15789 opt4 11731 12100 12469 12838 13945 14314 14683 15052 15421 15790 opt5 11732 12101 12470 12839 13946 14315 14684 15053 15422 15791 opt6 11733 12102 12471 12840 13947 14316 14685 15054 15423 15792 opt11 11734 12103 12472 12841 13948 14317 14686 15055 15424 15793

In Table 4A and 4B, Columns K to V represent specific suitable constructs of the invention derived from RSV Fusion (F) protein, wherein Column K provides suitable sequences for F_DSCav1_mut0, Column L provides suitable sequences for F-del_DSCav1_mut0, Column M provides suitable sequences for F_DSCav1_mut4, Column N provides suitable sequences for F-del_DSCav1_mut4, Column O provides suitable sequences for F_DSCav1_mut5, Column P provides suitable sequences for F-del_DSCav1_mut5, Column Q provides suitable sequences for F_DSCav1_mut6, Column R provides suitable sequences for F-del_DSCav1_mut6, Column S provides suitable sequences for F_DSCav1_mut7, Column T provides suitable sequences for F-del_DSCav1_mut7, Column U provides suitable sequences for F_DSCav1_mut8, Column V provides suitable sequences for F-del_DSCav1_mut8. The specific protein SEQ ID NOs as provided in the sequence listing are in row 2 (“PRT”). The SEQ ID NOs of corresponding codon modified coding sequences for each protein construct are provided in row 3 to row 9 (“opt1”, “opt2”, “opt3”, “opt4”, “opt5”, “opt6”, “opt11”). In Table 4A, coding sequences derived from HRSV(A2) are provided, in Table 4B coding sequences derived from HRSV(Memphis-37) are provided. Further information is provided in the <223> identifier for each of the respective SEQ ID NO in the sequence listing.

TABLE 4A Preferred coding sequences encoding RSV F (columns K-V), derived from HRSV(A2) K L M N O P Q R S T U V PRT 3850 4219 4588 4957 5326 5695 6064 6433 6802 7171 7540 7909 opt1 3851, 4220, 4589, 4958, 5327, 5696, 6065, 6434, 6803, 7172, 7541, 7910, 3852, 4221, 4590, 4959, 5328, 5697, 6066, 6435, 6804, 7173, 7542, 7911, 21367  21368  21375  21376  21377  21378  21379  21380  21381  21382  21383  21384  opt2 3853 4222 4591 4960 5329 5698 6067 6436 6805 7174 7543 7912 opt3 3854 4223 4592 4961 5330 5699 6068 6437 6806 7175 7544 7913 opt4 3855 4224 4593 4962 5331 5700 6069 6438 6807 7176 7545 7914 opt5 3856 4225 4594 4963 5332 5701 6070 6439 6808 7177 7546 7915 opt6 3857 4226 4595 4964 5333 5702 6071 6440 6809 7178 7547 7916 opt11 3858 4227 4596 4965 5334 5703 6072 6441 6810 7179 7548 7917

TABLE 4B Preferred coding sequences encoding RSV F (columns K-V) derived from HRSV(Memphis-37) K L M N O P Q R S T U V PRT 13202 13571 16154 16523 16892 17261 17630 17999 18368 18737 19106 19475 opt1 13203, 13572, 16155, 16524, 16893, 17262, 17631, 18000, 18369, 18738, 19107, 19476, 13204, 13573, 16156, 16525, 16894, 17263, 17632, 18001, 18370, 18739, 19108, 19477, 21393 21394 21401 21402 21403 21404 21405 21406 21407 21408 21409 21410 opt2 13205 13574 16157 16526 16895 17264 17633 18002 18371 18740 19109 19478 opt3 13206 13575 16158 16527 16896 17265 17634 18003 18372 18741 19110 19479 opt4 13207 13576 16159 16528 16897 17266 17635 18004 18373 18742 19111 19480 opt5 13208 13577 16160 16529 16898 17267 17636 18005 18374 18743 19112 19481 opt6 13209 13578 16161 16530 16899 17268 17637 18006 18375 18744 19113 19482 opt11 13210 13579 16162 16531 16900 17269 17638 18007 18376 18745 19114 19483

In embodiments, the artificial RNA of the first aspect is monocistronic, bicistronic, or multicistronic.

In preferred embodiments, the artificial RNA of the invention is monocistronic.

The term “monocistronic nucleic acid” or “monocistronic nucleic acid” will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to an artificial RNA that comprises only one coding sequences as defined herein. The terms “bicistronic nucleic acid, multicistronic nucleic acid” or “monocistronic RNA” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to an artificial RNA that may have two (bicistronic) or even more (multicistronic) coding sequences.

In embodiments, the artificial RNA of the invention is monocistronic and the coding sequence of said monocistronic artificial RNA encodes at least two different antigenic peptides or proteins derived from RSV F as defined herein, or a fragment or variant thereof. Accordingly, the at least one coding sequence of the monocistronic artificial RNA may encode at least two, three, four, five, six, seven, eight and more antigenic peptides or proteins derived from a RSV, preferably a RSV F as defined herein linked with or without an amino acid linker sequence, wherein said linker sequence can comprise rigid linkers, flexible linkers, cleavable linkers (e.g. self-cleaving peptides) as defined above, or a combination thereof (herein referred to as “multi-antigen-constructs/nucleic acid”).

In embodiments, the artificial RNA of the invention is bicistronic or multicistronic and comprises at least two coding sequences, wherein the at least two coding sequences encode two or more different antigenic peptides or proteins derived from RSV, preferably RSV F as defined herein, or a fragment or variant of any of these. Accordingly, the coding sequences in a bicistronic or multicistronic artificial RNA suitably encodes distinct antigenic proteins or peptides as defined herein or a fragment or variant thereof. Preferably, the coding sequences in said bicistronic or multicistronic artificial RNA may be separated by at least one IRES (internal ribosomal entry site) sequence. Thus, the term “encoding two or more antigenic peptides or proteins” may mean, without being limited thereto, that the bicistronic or multicistronic artificial RNA encodes e.g. at least two, three, four, five, six or more (preferably different) antigenic peptides or proteins of different RSV or their fragments or variants within the definitions provided herein. Alternatively, the bicistronic or multicistronic artificial RNA may encode e.g. at least two, three, four, five, six or more (preferably different) antigenic peptides or proteins derived from the same RSV or fragments or variants within the definitions provided herein. In that context, suitable IRES sequences may be selected from the list of nucleic acid sequences according to SEQ ID NOs: 1566-1662 of the patent application WO2017/081082, or fragments or variants of these sequences. In this context, the disclosure of WO2017/081082 relating to IRES sequences is herewith incorporated by reference.

It has to be understood that in the context of the invention, certain combinations of coding sequences may be generated by any combination of monocistronic, bicistronic and multicistronic artificial nucleic acids and/or multi-antigen-constructs/nucleic acid to obtain a nucleic acid composition encoding multiple antigenic peptides or proteins as defined herein.

Preferably, the artificial RNA comprising at least one coding sequence as defined herein typically comprises a length of about 50 to about 20000, or 500 to about 20000 nucleotides, or about 500 to about 20000 nucleotides, or about 500 to about 10000 nucleotides, or of about 1000 to about 10000 nucleotides, or preferably of about 1000 to about 5000 nucleotides, or even more preferably of about 1000 to about 2500 nucleotides.

According to preferred embodiments, the artificial RNA of the first aspect may be an mRNA, a self-replicating RNA, a circular RNA, or a replicon RNA.

In embodiments, the artificial RNA is a circular RNA. As used herein, “circular RNA” or “circRNAs” has to be understood as a circular polynucleotide that can encode at least one antigenic peptide or protein as defined herein. Accordingly, in preferred embodiments, said circular RNA comprises at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV or a fragment or variant thereof as defined herein. The production of circRNAs can be performed using various methods provided in the art. For example, U.S. Pat. No. 6,210,931 teaches a method of synthesizing circRNAs by inserting DNA fragments into a plasmid containing sequences having the capability of spontaneous cleavage and self-circularization. U.S. Pat. No. 5,773,244 teaches producing circRNAs by making a DNA construct encoding an RNA cyclase ribozyme, expressing the DNA construct as an RNA, and then allowing the RNA to self-splice, which produces a circRNA free from intron in vitro. WO1992/001813 teaches a process of making single strand circular nucleic acids by synthesizing a linear polynucleotide, combining the linear nucleotide with a complementary linking oligonucleotide under hybridization conditions, and ligating the linear polynucleotide. The person skilled in the art may also use methods provided in WO2015/034925 or WO2016/011222 to produce circular RNA. Accordingly, methods for producing circular RNA as provided in U.S. Pat. Nos. 6,210,931, 5,773,244, WO1992/001813, WO2015/034925 and WO2016/011222 are incorporated herewith by reference.

In embodiments, the artificial RNA is a replicon RNA. The term “replicon RNA” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to be optimized self-replicating artificial RNA constructs. Such constructs include replication elements (replicase) derived from alphaviruses and the substitution of the structural virus proteins with the artificial nucleic acid of interest (in the context of the invention, an artificial nucleic acid comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV. Alternatively, the replicase may be provided on an independent construct comprising a replicase RNA sequence derived from e.g. Semliki forest virus (SFV), Sindbis virus (SIN), Venezuelan equine Encephalitis virus (VEE), Ross-River virus (RRV), or other viruses belonging to the alphavirus family. Downstream of the replicase lies a sub-genomic promoter that controls replication of the artificial nucleic acid of the invention, i.e. an artificial nucleic acid comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV.

In preferred embodiments the artificial RNA of the first aspect is an mRNA.

The terms “RNA” and “mRNA” will be recognized and understood by the person of ordinary skill in the art, and are for example intended to be a ribonucleic acid molecule, i.e. a polymer consisting of nucleotides. These nucleotides are usually adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers which are connected to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific succession of the monomers is called the RNA-sequence. The mRNA (messenger RNA) usually provides the nucleotide sequence that may be translated into an amino-acid sequence of a particular peptide or protein.

The artificial RNA, preferably the mRNA of the invention may be prepared using any method known in the art, including chemical synthesis such as e.g. solid phase RNA synthesis, as well as in vitro methods, such as RNA in vitro transcription reactions.

In a preferred embodiment, the artificial RNA, preferably the mRNA is obtained by RNA in vitro transcription.

Accordingly, the RNA of the invention is preferably an in vitro transcribed RNA.

The terms “RNA in vitro transcription” or “in vitro transcription” relate to a process wherein RNA is synthesized in a cell-free system (in vitro). RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which according to the present invention is a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are the T7, T3, SP6, or Syn5 RNA polymerases. In a preferred embodiment of the present invention the DNA template is linearized with a suitable restriction enzyme, before it is subjected to RNA in vitro transcription.

Reagents used in RNA in vitro transcription typically include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); optionally, a cap analogue as defined herein (e.g. m7G(5′)ppp(5′)G (m7G, m7G(5′)ppp(5′)(2′OMeG)pG or m7G(5′)ppp(5′)(2′OMeA)pG)); optionally, further modified nucleotides as defined herein; a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g. T7, T3, SP6, or Syn5 RNA polymerase); optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to degrade pyrophosphate, which may inhibit RNA in vitro transcription; MgCl2, which supplies Mg2+ ions as a co-factor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and/or polyamines such as spermidine at optimal concentrations, e.g. a buffer system comprising TRIS-Citrate as disclosed in WO2017/109161.

In embodiments, the nucleotide mixture used in RNA in vitro transcription may additionally contain modified nucleotides as defined herein. In that context, preferred modified nucleotides comprise pseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and/or 5-methoxyuridine.

In preferred embodiments, the nucleotide mixture (i.e. the fraction of each nucleotide in the mixture) used for RNA in vitro transcription reactions may be optimized for the given RNA sequence, preferably as described WO2015/188933.

In embodiment where more than one different artificial RNA as defined herein has to be produced, e.g. where 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different artificial RNAs have to be produced (e.g. encoding different RSV F antigens, or e.g. a combination of RSV F and RSV G; see second aspect), procedures as described in WO2017/109134 may be suitably used.

In the context of RNA vaccine production, it may be required to provide GMP-grade RNA. GMP-grade RNA may be produced using a manufacturing process approved by regulatory authorities. Accordingly, in a particularly preferred embodiment, RNA production is performed under current good manufacturing practice (GMP), implementing various quality control steps on DNA and RNA level, preferably according to WO2016/180430. In preferred embodiments, the RNA of the invention is a GMP-grade RNA, particularly a GMP-grade mRNA.

The obtained RNA products are preferably purified using PureMessenger® (CureVac, Tubingen, Germany; RP-HPLC according to WO2008/077592) and/or tangential flow filtration (as described in WO2016/193206).

In a further preferred embodiment, the RNA, particularly the purified RNA, is lyophilized according to WO2016/165831 or WO2011/069586 to yield a temperature stable dried artificial RNA (powder) as defined herein. The RNA of the invention, particularly the purified RNA may also be dried using spray-drying or spray-freeze drying according to WO2016/184575 or WO2016184576 to yield a temperature stable RNA (powder) as defined herein. Accordingly, in the context of manufacturing and purifying RNA, the disclosures of WO2017/109161, WO2015/188933, WO2016/180430, WO2008/077592, WO2016/193206, WO2016/165831, WO2011/069586, WO2016/184575, and WO2016/184576 are incorporated herewith by reference.

Accordingly, in preferred embodiments, the RNA is a dried RNA, particularly a dried mRNA.

The term “dried RNA” as used herein has to be understood as RNA that has been lyophilized, or spray-dried, or spray-freeze dried as defined above to obtain a temperature stable dried RNA (powder).

In preferred embodiments, the artificial RNA of the invention is a purified RNA, particularly purified mRNA.

The term “purified RNA” or “purified mRNA” as used herein has to be understood as RNA which has a higher purity after certain purification steps (e.g. HPLC, TFF, Oligo d(T) purification, precipitation steps) than the starting material (e.g. in vitro transcribed RNA). Typical impurities that are essentially not present in purified RNA comprise peptides or proteins (e.g. enzymes derived from DNA dependent RNA in vitro transcription, e.g. RNA polymerases, RNases, pyrophosphatase, restriction endonuclease, DNase), spermidine, BSA, abortive RNA sequences, RNA fragments (short double stranded RNA fragments, abortive sequences etc.), free nucleotides (modified nucleotides, conventional NTPs, cap analogue), template DNA fragments, buffer components (HEPES, TRIS, MgCl2) etc. Other potential impurities that may be derived from e.g. fermentation procedures comprise bacterial impurities (bioburden, bacterial DNA) or impurities derived from purification procedures (organic solvents etc.). Accordingly, it is desirable in this regard for the “degree of RNA purity” to be as close as possible to 100%. It is also desirable for the degree of RNA purity that the amount of full-length RNA transcripts is as close as possible to 100%. Accordingly “purified RNA” as used herein has a degree of purity of more than 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and most favorably 99% or more. The degree of purity may for example be determined by an analytical HPLC, wherein the percentages provided above correspond to the ratio between the area of the peak for the target RNA and the total area of all peaks representing the by-products. Alternatively, the degree of purity may for example be determined by an analytical agarose gel electrophoresis or capillary gel electrophoresis.

It has to be understood that “dried RNA” as defined herein and “purified RNA” as defined herein or “GMP-grade mRNA” as defined herein may have superior stability characteristics (in vitro, in vivo) and improved efficiency (e.g. better translatability of the mRNA in vivo) and are therefore particularly suitable in the context of the invention. Moreover, “dried RNA” as defined herein and “purified RNA” as defined herein or “GMP-grade mRNA” may be particularly suitable for medical use as defined herein.

The artificial RNA may suitably be modified by the addition of a 5′-cap structure, which preferably stabilizes the nucleic acid as described herein. Accordingly, in preferred embodiments, the artificial RNA of the first aspect comprises a 5′-cap structure, preferably m7G, cap0 (e.g. m7G(5′)ppp(5′)G), cap1 (e.g. m7G(5′)ppp(5′)(2′OMeG) or m7G(5′)ppp(5′)(2′OMeA)), cap2, a modified cap0, or a modified cap1 structure (generated using a cap analogue as defined below).

The term “5′-cap structure” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a modified nucleotide (cap analogue), particularly a guanine nucleotide, added to the 5′-end of an RNA molecule, e.g. an mRNA molecule. Preferably, the 5′-cap is added using a 5-5-triphosphate linkage (also named m7GpppN). Further examples of 5′-cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4′,5′ methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3-inverted nucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediol phosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety.

Further 5′-cap structures which may be suitable in the context of the present invention are cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

A 5′-cap (cap0 or cap1) structure may also be formed in chemical RNA synthesis or, preferably, RNA in vitro transcription (co-transcriptional capping) using cap analogues.

The term “cap analogue” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a non-polymerizable di-nucleotide that has cap functionality in that it facilitates translation or localization, and/or prevents degradation of a nucleic acid molecule, particularly of an RNA molecule, when incorporated at the 5′-end of the nucleic acid molecule. Non-polymerizable means that the cap analogue will be incorporated only at the 5′-terminus because it does not have a 5′ triphosphate and therefore cannot be extended in the 3′-direction by a template-dependent polymerase, particularly, by template-dependent RNA polymerase. Examples of cap analogues include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogues (e.g. GpppG); dimethylated cap analogue (e.g. m2,7GpppG), trimethylated cap analogue (e.g. m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g. m7Gpppm7G), or anti reverse cap analogues (e.g. ARCA; m7,2′OmeGpppG, m7,2′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives). Further cap analogues have been described previously (WO2008/016473, WO2008/157688, WO2009/149253, WO2011/015347, and WO2013/059475). Further suitable cap analogons in that context are described in WO2017/066793, WO2017/066781, WO2017/066791, WO2017/066789, WO2017/053297, WO2017/066782, WO2018075827 and WO2017/066797 wherein the disclosures referring to cap analogues are incorporated herewith by reference.

The 5′-cap structure may suitably be added co-transcriptionally using cap-analogues as defined herein in an RNA in vitro transcription reaction as defined herein.

In preferred embodiments, a modified cap1 structure is generated using a cap analogue as disclosed in WO2017/053297, WO2017/066793, WO2017/066781, WO2017/066791, WO2017/066789, WO2017/066782, WO2018075827 and WO2017/066797. In particular, any cap structures derivable from the structure disclosed in claim 1-5 of WO2017/053297 may be suitably used to co-transcriptionally generate a modified cap1 structure. Further, any cap structures derivable from the structure defined in claim 1 or claim 21 of WO2018075827 may be suitably used to co-transcriptionally generate a modified cap1 structure.

Preferred cap-analogues are the di-nucleotide cap analogues m7G(5′)ppp(5′)G (m7G) or 3 O-Me-m7G(5′)ppp(5′)G to co-transcriptionally generate cap0 structures. Particularly preferred cap-analogues are the tri-nucleotide cap analogues m7G(5′)ppp(5′)(2′OMeA)pG or m7G(5′)ppp(5′)(2′OMeG)pG to co-transcriptionally generate cap1 structures.

In that context, it is preferred that the RNA of the invention comprises a Cap1 structure as defined above, which preferably result in an increased protein expression through e.g. high capping efficiencies and increased translation efficiencies. Further suitably, the RNA of the invention comprising a Cap1 structure displays a decreased stimulation of the innate immune system as compared to Cap0 constructs of the same nucleic acid sequence. The person of ordinary skill knows how to determine translation efficiencies, capping degree, and immune stimulation.

In a particularly preferred embodiment, the artificial RNA of the first aspect of the invention comprises a cap1 structure, wherein said cap1 structure may be formed enzymatically or co-transcriptionally (e.g. using m7G(5′)ppp(5′)(2′OMeA)pG or m7G(5′)ppp(5′)(2′OMeG)pG analogues).

In preferred embodiments, the artificial RNA of the first aspect comprises an m7G(5′)ppp(5′)(2′OMeA)pG cap structure. In such embodiments, the coding RNA comprises a 5′ terminal m7G cap, and an additional methylation of the ribose of the adjacent nucleotide of m7GpppN, in that case, a 2′O methylated adenosine.

In other preferred embodiments, the artificial RNA of the first aspect comprises an m7G(5′)ppp(5′)(2′OMeG)pG cap structure. In such embodiments, the coding RNA comprises a 5′ terminal m7G cap, and an additional methylation of the ribose of the adjacent nucleotide, in that case, a 2′O methylated guanosine.

Accordingly, whenever reference is made to suitable RNA or mRNA sequences in the context of the invention, the first nucleotide of said RNA or mRNA sequence, that is the nucleotide downstream of the m7G(5′)ppp structure, may be a 2′O methylated guanosine or a 2′O methylated adenosine.

Accordingly, in other embodiments, the artificial RNA of the invention may comprise a 5′-cap sequence element according to SEQ ID NOs: 43 or 21321, or a fragment or variant thereof.

In other embodiments, the 5′-cap structure is added via enzymatic capping using capping enzymes (e.g. vaccinia virus capping enzymes, commercially available capping kits) to generate cap or cap1 or cap2 structures. In other embodiments, the 5′-cap structure (cap0, cap1) is added via enzymatic capping using immobilized capping enzymes, e.g. using a capping reactor (WO2016/193226).

In preferred embodiments, the artificial RNA of the invention comprises at least one poly(A) sequence, preferably comprising 30 to 150 adenosine nucleotides.

In preferred embodiments, the poly(A) sequence, suitable located at the 3′ terminus, comprises 10 to 500 adenosine nucleotides, 10 to 200 adenosine nucleotides, 40 to 200 adenosine nucleotides or 40 to 150 adenosine nucleotides. In a particularly preferred embodiment, the poly(A) sequence comprises about 64 adenosine nucleotides. In further particularly preferred embodiments, the poly(A) sequence comprises about 75 adenosine nucleotides. In further particularly preferred embodiments, the poly(A) sequence comprises about 100 adenosine nucleotides.

The terms “poly(A) sequence”, “poly(A) tail” or “3′-poly(A) tail” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to be a sequence of adenosine nucleotides, typically located at the 3′-end of an RNA, of up to about 1000 adenosine nucleotides. Preferably, said poly(A) sequence is essentially homopolymeric, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotides has essentially the length of 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine nucleotide, e.g. a poly(A) sequence of e.g. 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide different from an adenosine nucleotide).

In the context of the present invention, a poly(A) sequence may be located within an mRNA or any other nucleic acid molecule, such as, e.g., in a DNA vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription said DNA vector.

Preferably, the poly(A) sequence of the artificial RNA is obtained from a DNA template during RNA in vitro transcription. In other embodiments, the poly(A) sequence is obtained in vitro by common methods of chemical synthesis without being necessarily transcribed from a DNA template. In other embodiments, poly(A) sequences are generated by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using commercially available polyadenylation kits and corresponding protocols known in the art, or alternatively, by using immobilized poly(A)polymerases e.g. using a polyadenylation reactor (as described in WO2016/174271).

In embodiments, the artificial RNA of the invention may contain a poly(A) sequence derived from a vector and may comprise at least one additional poly(A) sequence generated by enzymatic polyadenylation, e.g. as described in WO2016/091391.

In preferred embodiments, the artificial RNA of the invention comprises at least one poly(C) sequence, preferably comprising 10 to 40 cytosine nucleotides.

In preferred embodiments, the poly(C) sequence, suitable located at the 3′ terminus, comprises 10 to 200 cytosine nucleotides, 10 to 100 cytosine nucleotides, 20 to 70 cytosine nucleotides, 20 to 60 cytosine nucleotides, or 10 to 40 cytosine nucleotides. In a particularly preferred embodiment, the poly(C) sequence comprises about cytosine nucleotides.

The term “poly(C) sequence” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to be a sequence of cytosine nucleotides, typically located at the 3′-end of an RNA, of up to about 200 cytosine nucleotides. In the context of the present invention, a poly(C) sequence may be located within an mRNA or any other nucleic acid molecule, such as, e.g., in a DNA vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription of the vector.

Preferably, the poly(C) sequence in the RNA sequence of the present invention is derived from a DNA template by RNA in vitro transcription. In other embodiments, the poly(C) sequence is obtained in vitro by common methods of chemical synthesis without being necessarily transcribed from a DNA template.

In other embodiments, the artificial RNA of the invention does not comprises a poly(C) sequence as defined herein.

In preferred embodiments, the artificial RNA of the first aspect comprises at least one histone stem-loop.

The term “histone stem-loop” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to nucleic acid sequences that are predominantly found in histone mRNAs. Exemplary histone stem-loop sequences are described in Lopez et al. (Davila Lopez et al, (2008), RNA, 14(1)). The stem-loops in histone pre-mRNAs are typically followed by a purine-rich sequence known as the histone downstream element (HDE). These pre-mRNAs are processed in the nucleus by a single endonucleolytic cleavage approximately 5 nucleotides downstream of the stem-loop, catalysed by the U7 snRNP through base pairing of the U7 snRNA with the HDE.

Histone stem-loop sequences/structures may suitably be selected from histone stem-loop sequences as disclosed in WO2012/019780, the disclosure relating to histone stem-loop sequences/structures incorporated herewith by reference. A histone stem-loop sequence that may be used within the present invention may preferably be derived from formulae (I) or (II) of the patent application WO2012/019780. According to a further preferred embodiment the RNA as defined herein may comprise at least one histone stem-loop sequence derived from at least one of the specific formulae (Ia) or (IIa) of the patent application WO2012/019780.

In particularly preferred embodiment, the artificial RNA of the invention comprises at least one histone stem-loop, wherein said histone stem-loop comprises a nucleic acid sequence according to SEQ ID NO: 39 or 40 or a fragments or variant thereof.

In other embodiments, the artificial RNA of the first aspect does not comprises a histone stem-loop as defined herein.

In further embodiments, the artificial RNA of the invention comprises a 3′-terminal sequence element. Said 3′-terminal sequence element has to be understood as a sequence element comprising a poly(A)sequence and a histone-stem-loop sequence, wherein said sequence element is located at the 3′ terminus of the artificial RNA of the invention.

In other embodiments, the artificial RNA of the invention may comprise a 3′-terminal sequence element according to SEQ ID NOs: 44-63 or 21322-21328 or a fragment or variant thereof.

In preferred embodiments, the artificial RNA of the invention comprises at least one pseudouridine (ψ) modified coding sequence.

Accordingly, in preferred embodiments, the artificial RNA of the invention, or the at least one coding sequence, comprises a nucleic acid sequence wherein at least one or more than one, preferably wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides.

In further preferred embodiments, the artificial RNA of the invention comprises at least one N1-methylpseudouridine (m1ψ) modified coding sequence.

Accordingly, in preferred embodiments, the artificial RNA of the invention, or the at least one coding sequence, comprises a nucleic acid sequence wherein at least one or more than one, preferably wherein all uracil nucleotides are replaced by N1-methylpseudouridine (m1P) nucleotides

In preferred embodiments of the first aspect, the artificial RNA, preferably mRNA comprises preferably in 5′- to 3-direction the following elements a)-i):

-   a) 5′-cap structure, preferably as specified herein; -   b) optionally, 5′-UTR as specified herein, preferably at least one     selected from SEQ ID NOs: 1-22; -   c) a ribosome binding site, preferably as specified herein -   d) at least one coding sequence as specified herein, preferably as     specified in Table 3 and Table 4; -   e) 3′-UTR as specified herein, preferably at least one selected from     SEQ ID NOs: 23-38; -   f) optionally, a poly(A) sequence, preferably as specified herein; -   g) optionally, a poly(C) sequence, preferably as specified herein; -   h) optionally, a histone stem-loop, preferably as specified herein; -   i) optionally, a 3′-terminal sequence element as specified herein,     preferably according to according to SEQ ID NOs: 44-63, or     21322-21328; and -   wherein optionally at least one or more than one, preferably wherein     all uracil nucleotides are replaced by pseudouridine (L) nucleotides     or N1-methylpseudouridine (m1ψ) nucleotides.

In further preferred embodiments of the first aspect, the artificial RNA, preferably mRNA comprises the following elements preferably in 5′- to 3-direction:

-   a) 5′-cap structure, preferably as specified herein, most preferably     a Cap1 structure; -   b) a 3′-UTR and a 5′-UTR element according to a-1, a-4, c-1, e-4,     g-2, i-2, or i-3 as specified herein; -   c) a ribosome binding site, preferably as specified herein; -   d) at least one coding sequence as specified herein, wherein said     coding sequence is located between said 5′-UTR and said 3′-UTR,     preferably downstream of said 5′-UTR and upstream of said 3′-UTR,     wherein the coding sequence is preferably selected from any one     specified in Table 3 and Table 4; -   e) optionally, a poly(A) sequence, preferably as specified herein; -   f) optionally, poly(C) sequence, preferably as specified herein; -   g) optionally, histone stem-loop, preferably as specified herein; -   h) optionally, a 3′-terminal sequence element as specified herein,     preferably according to according to SEQ ID NOs: 44-63, 21322-21328,     and -   wherein optionally at least one or more than one, preferably wherein     all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides     or N1-methylpseudouridine (m1ψ) nucleotides.

In further preferred embodiments of the first aspect, the artificial RNA, preferably mRNA comprises the following elements preferably in 5′- to 3-direction:

-   a) 5′-cap structure, preferably as specified herein, most preferably     a Cap1 structure; -   b) a 3′-UTR and a 5′-UTR element according to a-1 or i-3 as     specified herein; -   c) a ribosome binding site, preferably as specified herein -   d) at least one coding sequence as specified herein, wherein said     coding sequence is located between said 5′-UTR and said 3′-UTR,     preferably downstream of said 5′-UTR and upstream of said 3′-UTR,     wherein the coding sequence is preferably selected from any one     specified in Table 3 and Table 4; -   e) optionally, a histone stem-loop, preferably as specified herein; -   f) a poly(A) sequence, preferably comprising about 100 adenosine     nucleotides; -   g) optionally, a 3′-terminal sequence element as specified herein,     preferably according to according to SEQ ID NOs: 21322-21328, and -   wherein optionally at least one or more than one, preferably wherein     all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides     or N1-methylpseudouridine (m1γ) nucleotides.

In further preferred embodiments of the first aspect, the artificial RNA, preferably mRNA comprises the following elements in 5′- to 3′-direction:

-   a) 5′-cap structure, preferably as specified herein, most preferably     a Cap1 structure; -   b) a 3′-UTR and a 5′-UTR element according to a-1, a-4, c-1, e-4,     g-2, i-2, or i-3 as specified herein; -   c) a ribosome binding site, preferably as specified herein; -   d) at least one coding sequence as specified herein, wherein said     coding region is located between said 5′-UTR and said 3′-UTR,     preferably downstream of said 5′-UTR and upstream of said 3′-UTR,     wherein the coding sequence is preferably selected from any one of     SEQ ID NOs: 69-77, 484-492, 899-906, 1268-1275, 1637-1644,     2006-2013, 2375-2382, 2744-2751, 3113-3120, 3482-3489, 3851-3858,     4220-4227, 4589-4596, 4958-4965, 5327-5334, 5696-5703, 6065-6072,     6434-6441, 6803-6810, 7172-7179, 7541-7548, 7910-7917, 11727-11734,     12096-12103, 12465-12472, 12834-12841, 13941-13948, 14310-14317,     14679-14686, 15048-15055, 15417-15424, 15786-15793, 13203-13210,     13572-13579, 16155-16162, 16524-16531, 16893-16900, 17262-17269,     17631-17638, 18000-18007, 18369-18376, 18738-18745, 19107-19114,     19476-19483, 21363-21384, 21389-21410 (or fragments or variants     thereof); -   e) a poly(A) sequence comprising about 64 adenosine; -   f) a poly(C) sequence comprising about 30 cysteines; -   g) a histone stem-loop according to SEQ ID NO: 39 or 40, and -   wherein optionally at least one or more than one, preferably wherein     all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides     or N1-methylpseudouridine (m1ψ) nucleotides.

In further preferred embodiments of the first aspect, the artificial RNA, preferably mRNA comprises the following elements in 5′- to 3′-direction:

-   a) 5′-cap structure, preferably as specified herein, most preferably     a Cap1 structure; -   b) a 3′-UTR and a 5′-UTR element according to a-1, a-4, c-1, e-4,     g-2, i-2, or i-3 as specified herein; -   c) a ribosome binding site, preferably as specified herein; -   d) at least one coding sequence as specified herein, wherein said     coding region is located between said 5′-UTR and said 3′-UTR,     preferably downstream of said 5′-UTR and upstream of said 3′-UTR,     wherein the coding sequence is preferably selected from any one of     SEQ ID NOs: 69-77, 484-492, 899-906, 1268-1275, 1637-1644,     2006-2013, 2375-2382, 2744-2751, 3113-3120, 3482-3489, 3851-3858,     4220-4227, 4589-4596, 4958-4965, 5327-5334, 5696-5703, 6065-6072,     6434-6441, 6803-6810, 7172-7179, 7541-7548, 7910-7917, 11727-11734,     12096-12103, 12465-12472, 12834-12841, 13941-13948, 14310-14317,     14679-14686, 15048-15055, 15417-15424, 15786-15793, 13203-13210,     13572-13579, 16155-16162, 16524-16531, 16893-16900, 17262-17269,     17631-17638, 18000-18007, 18369-18376, 18738-18745, 19107-19114,     19476-19483, 21363-21384, 21389-21410 (or fragments or variants     thereof); -   e) a poly(A) sequence comprising about 64 adenosine; -   f) a histone stem-loop according to SEQ ID NO: 39 or 40, and -   wherein optionally at least one or more than one, preferably wherein     all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides     or N1-methylpseudouridine (m1ψ) nucleotides.

Preferred RSV polypeptide, nucleic acid, and mRNA sequences are provided in Table 5A, 5B and Table 6A, 6B.

In Table 5A and 5B, the protein designs are indicated in row 1. Therein, Columns A to J represent specific suitable constructs of the invention derived from RSV Fusion (F) protein, wherein Column A provides suitable sequences for F0, Column B provides suitable sequences for F-del, Column C provides suitable sequences for F0_DSCav1, Column D provides suitable sequences for F-del_DSCav1, Column E provides suitable sequences for F_DSCav1_mut1, Column F provides suitable sequences for F-del_DSCav1_mut1, Column G provides suitable sequences for F_DSCav1_mut2, Column H provides suitable sequences for F-del_DSCav1_mut2, Column I provides suitable sequences for F_DSCav1_mut3, Column J provides suitable sequences for F-del_DSCav1_mut3.

The protein designs are indicated in row 1 (Columns A to J), the specific protein SEQ ID NOs as provided in the sequence listing are in row 2 (“protein”). The SEQ ID NOs of corresponding coding sequences for each protein construct are provided in row 3 (“cds”, compare with Table 3 for different cds optimizations). Further information e.g. regarding the type of codon modified coding sequence (opt1, opt2, opt3, opt4, opt5, opt6, opt11 etc.) is provided in the <223> identifier of the respective SEQ ID NO in the sequence listing and in Table 3. The SEQ ID NOs of corresponding mRNA constructs comprising said coding sequences and suitable 3′-UTRs and 5′-UTRs according to the invention are provided in rows 4 to 47 (mRNA designs a-1 to i-3 as specified herein). In Table 5A, mRNA sequences derived from HRSV(A2) are provided, in Table 5B mRNA sequences derived from HRSV(Memphis-37) are provided. Further information e.g. regarding the type of coding sequence (wt, opt1, opt2, opt3, opt4, opt5, opt6, opt11 etc.) comprised in the mRNA constructs is provided in the <223> identifier of the respective SEQ ID NO in the sequence listing.

TABLE 5A Preferred mRNA constructs encoding RSV F (columns A-J), derived from HRSV(A2) 1 A B C D E F G H I J 2 Protein 68 483 898 1267 1636 2005 2374 2743 3112 3481 3 cds 69-77, 484- 899- 1268- 1637- 2006- 2375- 2744- 3113- 3482- 21363 492, 906, 1275, 1644, 2013, 2382, 2751, 3120, 3489, 21364 21365 21366 21369 21370 21371 21372 21373 21374 4 mRNA 78-86, 493- 907- 1276- 1645- 2014- 2383- 2752- 3121- 3490- Design a-1 21415- 501, 914, 1283, 1652, 2021, 2390, 2759, 3128, 3497, 21417, 21418- 21421- 21424- 21433- 21436- 21439- 21442- 21445- 21448- 21561- 21420, 21423, 21426, 21435, 21438, 21441, 21444, 21447, 21450, 21563 21564- 21567- 21570- 21579- 21582- 21585- 21588- 21591- 21594- 21566 21569 21572 21581 21584 21587 21590 21593 21596 5 mRNA 87-95 502- 915- 1284- 1653- 2022- 2391- 2760- 3129- 3498- Design a-2 510 922 1291 1660 2029 2398 2767 3136 3505 6 mRNA  96-104 511- 923- 1292- 1661- 2030- 2399- 2768- 3137- 3506- Design a-3 519 930 1299 1668 2037 2406 2775 3144 3513 7 mRNA 105-113 520- 931- 1300- 1669- 2038- 2407- 2776- 3145- 3514- Design a-4 528 938 1307 1676 2045 2414 2783 3152 3521 8 mRNA 114-122 529- 939- 1308- 1677- 2046- 2415- 2784- 3153- 3522- Design a-5 537 946 1315 1684 2053 2422 2791 3160 3529 9 mRNA 123-131 538- 947- 1316- 1685- 2054- 2423- 2792- 3161- 3530- Design b-1 546 954 1323 1692 2061 2430 2799 3168 3537 10 mRNA 132-140 547- 955- 1324- 1693- 2062- 2431- 2800- 3169- 3538- Design b-2 555 962 1331 1700 2069 2438 2807 3176 3545 11 mRNA 141-149 556- 963- 1332- 1701- 2070- 2439- 2808- 3177- 3546- Design b-3 564 970 1339 1708 2077 2446 2815 3184 3553 12 mRNA 150-158 565- 971- 1340- 1709- 2078- 2447- 2816- 3185- 3554- Design b-4 573 978 1347 1716 2085 2454 2823 3192 3561 13 mRNA 159-167 574- 979- 1348- 1717- 2086- 2455- 2824- 3193- 3562- Design b-5 582 986 1355 1724 2093 2462 2831 3200 3569 14 mRNA 168-176 583- 987- 1356- 1725- 2094- 2463- 2832- 3201- 3570- Design c-1 591 994 1363 1732 2101 2470 2839 3208 3577 15 mRNA 177-185 592- 995- 1364- 1733- 2102- 2471- 2840- 3209- 3578- Design c-2 600 1002 1371 1740 2109 2478 2847 3216 3585 16 mRNA 186-194 601- 1003- 1372- 1741- 2110- 2479- 2848- 3217- 3586- Design c-3 609 1010 1379 1748 2117 2486 2855 3224 3593 17 mRNA 195-203 610- 1011- 1380- 1749- 2118- 2487- 2856- 3225- 3594- Design c-4 618 1018 1387 1756 2125 2494 2863 3232 3601 18 mRNA 204-212 619- 1019- 1388- 1757- 2126- 2495- 2864- 3233- 3602- Design c-5 627 1026 1395 1764 2133 2502 2871 3240 3609 19 mRNA 213-221 628- 1027- 1396- 1765- 2134- 2503- 2872- 3241- 3610- Design d-1 636 1034 1403 1772 2141 2510 2879 3248 3617 20 mRNA 222-230 637- 1035- 1404- 1773- 2142- 2511- 2880- 3249- 3618- Design d-2 645 1042 1411 1780 2149 2518 2887 3256 3625 21 mRNA 231-239 646- 1043- 1412- 1781- 2150- 2519- 2888- 3257- 3626- Design d-3 654 1050 1419 1788 2157 2526 2895 3264 3633 22 mRNA 240-248 655- 1051- 1420- 1789- 2158- 2527- 2896- 3265- 3634- Design d-4 663 1058 1427 1796 2165 2534 2903 3272 3641 23 mRNA 249-257 664- 1059- 1428- 1797- 2166- 2535- 2904- 3273- 3642- Design d-5 672 1066 1435 1804 2173 2542 2911 3280 3649 24 mRNA 258-266 673- 1067- 1436- 1805- 2174- 2543- 2912- 3281- 3650- Design e-1 681 1074 1443 1812 2181 2550 2919 3288 3657 25 mRNA 267-275 682- 1075- 1444- 1813- 2182- 2551- 2920- 3289- 3658- Design e-2 690 1082 1451 1820 2189 2558 2927 3296 3665 26 mRNA 276-284 691- 1083- 1452- 1821- 2190- 2559- 2928- 3297- 3666- Design e-3 699 1090 1459 1828 2197 2566 2935 3304 3673 27 mRNA 285-293 700- 1091- 1460- 1829- 2198- 2567- 2936- 3305- 3674- Design e-4 708 1098 1467 1836 2205 2574 2943 3312 3681 28 mRNA 294-302 709- 1099- 1468- 1837- 2206- 2575- 2944- 3313- 3682- Design e-5 717 1106 1475 1844 2213 2582 2951 3320 3689 29 mRNA 303-311 718- 1107- 1476- 1845- 2214- 2583- 2952- 3321- 3690- Design e-6 726 1114 1483 1852 2221 2590 2959 3328 3697 30 mRNA 312-320 727- 1115- 1484- 1853- 2222- 2591- 2960- 3329- 3698- Design f-1 735 1122 1491 1860 2229 2598 2967 3336 3705 31 mRNA 321-329 736- 1123- 1492- 1861- 2230- 2599- 2968- 3337- 3706- Design f-2 744 1130 1499 1868 2237 2606 2975 3344 3713 32 mRNA 330-338 745- 1131- 1500- 1869- 2238- 2607- 2976- 3345- 3714- Design f-3 753 1138 1507 1876 2245 2614 2983 3352 3721 33 mRNA 339-347 754- 1139- 1508- 1877- 2246- 2615- 2984- 3353- 3722- Design f-4 762 1146 1515 1884 2253 2622 2991 3360 3729 34 mRNA 348-356 763- 1147- 1516- 1885- 2254- 2623- 2992- 3361- 3730- Design f-5 771 1154 1523 1892 2261 2630 2999 3368 3737 35 mRNA 357-365 772- 1155- 1524- 1893- 2262- 2631- 3000- 3369- 3738- Design g-1 780 1162 1531 1900 2269 2638 3007 3376 3745 36 mRNA 366-374 781- 1163- 1532- 1901- 2270- 2639- 3008- 3377- 3746- Design g-2 789 1170 1539 1908 2277 2646 3015 3384 3753 37 mRNA 375-383 790- 1171- 1540- 1909- 2278- 2647- 3016- 3385- 3754- Design g-3 798 1178 1547 1916 2285 2654 3023 3392 3761 38 mRNA 384-392 799- 1179- 1548- 1917- 2286- 2655- 3024- 3393- 3762- Design g-4 807 1186 1555 1924 2293 2662 3031 3400 3769 39 mRNA 393-401 808- 1187- 1556- 1925- 2294- 2663- 3032- 3401- 3770- Design g-5 816 1194 1563 1932 2301 2670 3039 3408 3777 40 mRNA 402-410 817- 1195- 1564- 1933- 2302- 2671- 3040- 3409- 3778- Design h-1 825 1202 1571 1940 2309 2678 3047 3416 3785 41 mRNA 411-419 826- 1203- 1572- 1941- 2310- 2679- 3048- 3417- 3786- Design h-2 834 1210 1579 1948 2317 2686 3055 3424 3793 42 mRNA 420-428 835- 1211- 1580- 1949- 2318- 2687- 3056- 3425- 3794- Design h-3 843 1218 1587 1956 2325 2694 3063 3432 3801 43 mRNA 429-437 844- 1219- 1588- 1957- 2326- 2695- 3064- 3433- 3802- Design h-4 852 1226 1595 1964 2333 2702 3071 3440 3809 44 mRNA 438-446 853- 1227- 1596- 1965- 2334- 2703- 3072- 3441- 3810- Design h-5 861 1234 1603 1972 2341 2710 3079 3448 3817 45 mRNA 447-455 862- 1235- 1604- 1973- 2342- 2711- 3080- 3449- 3818- Design i-1 870 1242 1611 1980 2349 2718 3087 3456 3825 46 mRNA 456-473 871- 1243- 1612- 1981- 2350- 2719- 3088- 3457- 3826- Design i-2 888 1258 1627 1996 2365 2734 3103 3472 3841 47 mRNA 474-482 889- 1259- 1628- 1997- 2366- 2735- 3104- 3473- 3842- Design i-3 897 1266 1635 2004 2373 2742 3111 3480 3849 48 mRNA 8278 Design i-4

TABLE 5B Preferred mRNA constructs encoding RSV F (columns A-J), derived from HRSV(Memphis-37) 1 A B C D E F G H I J 2 Protein 11726 12095 12464 12833 13940 14309 14678 15047 15416 15785 3 cds 11727- 12096- 12465- 12834- 13941- 14310- 14679- 15048- 15417- 15786- 11734, 12103, 12472, 12841, 13948, 14317, 14686, 15055, 15424, 15793, 21389 21390 21391 21392 21395 21396 21397 21398 21399 21400 4 mRNA 11735- 12104- 12473- 12842- 13949- 14318- 14687- 15056- 15425- 15794- Design a-1 11742, 12111, 12480, 12849, 13956, 14325, 14694, 15063, 15432, 15801, 21489, 21491, 21493- 21496- 21505- 21508- 21511- 21514- 21517- 21520- 21490, 21492, 21495, 21498, 21507, 21510, 21513, 21516, 21519, 21522, 21635, 21637, 21639- 21642- 21651- 21654- 21657- 21660- 21663- 21666- 21636 21638 21641 21644 21653 21656 21659 21662 21665 21668 5 mRNA 11743- 12112- 12481- 12850- 13957- 14326- 14695- 15064- 15433- 15802- Design a-2 11750 12119 12488 12857 13964 14333 14702 15071 15440 15809 6 mRNA 11751- 12120- 12489- 12858- 13965- 14334- 14703- 15072- 15441- 15810- Design a-3 11758 12127 12496 12865 13972 14341 14710 15079 15448 15817 7 mRNA 11759- 12128- 12497- 12866- 13973- 14342- 14711- 15080- 15449- 15818- Design a-4 11766 12135 12504 12873 13980 14349 14718 15087 15456 15825 8 mRNA 11767- 12136- 12505- 12874- 13981- 14350- 14719- 15088- 15457- 15826- Design a-5 11774 12143 12512 12881 13988 14357 14726 15095 15464 15833 9 mRNA 11775- 12144- 12513- 12882- 13989- 14358- 14727- 15096- 15465- 15834- Design b-1 11782 12151 12520 12889 13996 14365 14734 15103 15472 15841 10 mRNA 11783- 12152- 12521- 12890- 13997- 14366- 14735- 15104- 15473- 15842- Design b-2 11790 12159 12528 12897 14004 14373 14742 15111 15480 15849 11 mRNA 11791- 12160- 12529- 12898- 14005- 14374- 14743- 15112- 15481- 15850- Design b-3 11798 12167 12536 12905 14012 14381 14750 15119 15488 15857 12 mRNA 11799- 12168- 12537- 12906- 14013- 14382- 14751- 15120- 15489- 15858- Design b-4 11806 12175 12544 12913 14020 14389 14758 15127 15496 15865 13 mRNA 11807- 12176- 12545- 12914- 14021- 14390- 14759- 15128- 15497- 15866- Design b-5 11814 12183 12552 12921 14028 14397 14766 15135 15504 15873 14 mRNA 11815- 12184- 12553- 12922- 14029- 14398- 14767- 15136- 15505- 15874- Design c-1 11822 12191 12560 12929 14036 14405 14774 15143 15512 15881 15 mRNA 11823- 12192- 12561- 12930- 14037- 14406- 14775- 15144- 15513- 15882- Design c-2 11830 12199 12568 12937 14044 14413 14782 15151 15520 15889 16 mRNA 11831- 12200- 12569- 12938- 14045- 14414- 14783- 15152- 15521- 15890- Design c-3 11838 12207 12576 12945 14052 14421 14790 15159 15528 15897 17 mRNA 11839- 12208- 12577- 12946- 14053- 14422- 14791- 15160- 15529- 15898- Design c-4 11846 12215 12584 12953 14060 14429 14798 15167 15536 15905 18 mRNA 11847- 12216- 12585- 12954- 14061- 14430- 14799- 15168- 15537- 15906- Design c-5 11854 12223 12592 12961 14068 14437 14806 15175 15544 15913 19 mRNA 11855- 12224- 12593- 12962- 14069- 14438- 14807- 15176- 15545- 15914- Design d-1 11862 12231 12600 12969 14076 14445 14814 15183 15552 15921 20 mRNA 11863- 12232- 12601- 12970- 14077- 14446- 14815- 15184- 15553- 15922- Design d-2 11870 12239 12608 12977 14084 14453 14822 15191 15560 15929 21 mRNA 11871- 12240- 12609- 12978- 14085- 14454- 14823- 15192- 15561- 15930- Design d-3 11878 12247 12616 12985 14092 14461 14830 15199 15568 15937 22 mRNA 11879- 12248- 12617- 12986- 14093- 14462- 14831- 15200- 15569- 15938- Design d-4 11886 12255 12624 12993 14100 14469 14838 15207 15576 15945 23 mRNA 11887- 12256- 12625- 12994- 14101- 14470- 14839- 15208- 15577- 15946- Design d-5 11894 12263 12632 13001 14108 14477 14846 15215 15584 15953 24 mRNA 11895- 12264- 12633- 13002- 14109- 14478- 14847- 15216- 15585- 15954- Design e-1 11902 12271 12640 13009 14116 14485 14854 15223 15592 15961 25 mRNA 11903- 12272- 12641- 13010- 14117- 14486- 14855- 15224- 15593- 15962- Design e-2 11910 12279 12648 13017 14124 14493 14862 15231 15600 15969 26 mRNA 11911- 12280- 12649- 13018- 14125- 14494- 14863- 15232- 15601- 15970- Design e-3 11918 12287 12656 13025 14132 14501 14870 15239 15608 15977 27 mRNA 11919- 12288- 12657- 13026- 14133- 14502- 14871- 15240- 15609- 15978- Design e-4 11926 12295 12664 13033 14140 14509 14878 15247 15616 15985 28 mRNA 11927- 12296- 12665- 13034- 14141- 14510- 14879- 15248- 15617- 15986- Design e-5 11934 12303 12672 13041 14148 14517 14886 15255 15624 15993 29 mRNA 11935- 12304- 12673- 13042- 14149- 14518- 14887- 15256- 15625- 15994- Design e-6 11942 12311 12680 13049 14156 14525 14894 15263 15632 16001 30 mRNA 11943- 12312- 12681- 13050- 14157- 14526- 14895- 15264- 15633- 16002- Design f-1 11950 12319 12688 13057 14164 14533 14902 15271 15640 16009 31 mRNA 11951- 12320- 12689- 13058- 14165- 14534- 14903- 15272- 15641- 16010- Design f-2 11958 12327 12696 13065 14172 14541 14910 15279 15648 16017 32 mRNA 11959- 12328- 12697- 13066- 14173- 14542- 14911- 15280- 15649- 16018- Design f-3 11966 12335 12704 13073 14180 14549 14918 15287 15656 16025 33 mRNA 11967- 12336- 12705- 13074- 14181- 14550- 14919- 15288- 15657- 16026- Design f-4 11974 12343 12712 13081 14188 14557 14926 15295 15664 16033 34 mRNA 11975- 12344- 12713- 13082- 14189- 14558- 14927- 15296- 15665- 16034- Design f-5 11982 12351 12720 13089 14196 14565 14934 15303 15672 16041 35 mRNA 11983- 12352- 12721- 13090- 14197- 14566- 14935- 15304- 15673- 16042- Design g-1 11990 12359 12728 13097 14204 14573 14942 15311 15680 16049 36 mRNA 11991- 12360- 12729- 13098- 14205- 14574- 14943- 15312- 15681- 16050- Design g-2 11998 12367 12736 13105 14212 14581 14950 15319 15688 16057 37 mRNA 11999- 12368- 12737- 13106- 14213- 14582- 14951- 15320- 15689- 16058- Design g-3 12006 12375 12744 13113 14220 14589 14958 15327 15696 16065 38 mRNA 12007- 12376- 12745- 13114- 14221- 14590- 14959- 15328- 15697- 16066- Design g-4 12014 12383 12752 13121 14228 14597 14966 15335 15704 16073 39 mRNA 12015- 12384- 12753- 13122- 14229- 14598- 14967- 15336- 15705- 16074- Design g-5 12022 12391 12760 13129 14236 14605 14974 15343 15712 16081 40 mRNA 12023- 12392- 12761- 13130- 14237- 14606- 14975- 15344- 15713- 16082- Design h-1 12030 12399 12768 13137 14244 14613 14982 15351 15720 16089 41 mRNA 12031- 12400- 12769- 13138- 14245- 14614- 14983- 15352- 15721- 16090- Design h-2 12038 12407 12776 13145 14252 14621 14990 15359 15728 16097 42 mRNA 12039- 12408- 12777- 13146- 14253- 14622- 14991- 15360- 15729- 16098- Design h-3 12046 12415 12784 13153 14260 14629 14998 15367 15736 16105 43 mRNA 12047- 12416- 12785- 13154- 14261- 14630- 14999- 15368- 15737- 16106- Design h-4 12054 12423 12792 13161 14268 14637 15006 15375 15744 16113 44 mRNA 12055- 12424- 12793- 13162- 14269- 14638- 15007- 15376- 15745- 16114- Design h-5 12062 12431 12800 13169 14276 14645 15014 15383 15752 16121 45 mRNA 12063- 12432- 12801- 13170- 14277- 14646- 15015- 15384- 15753- 16122- Design i-1 12070 12439 12808 13177 14284 14653 15022 15391 15760 16129 46 mRNA 12071- 12440- 12809- 13178- 14285- 14654- 15023- 15392- 15761- 16130- Design i-2 12086 12455 12824 13193 14300 14669 15038 15407 15776 16145 47 mRNA 12087- 12456- 12825- 13194- 14301- 14670- 15039- 15408- 15777- 16146- Design i-3 12094 12463 12832 13201 14308 14677 15046 15415 15784 16153

In Table 6A and 6B, the protein designs are indicated in row 1. Therein, Columns K to V represent specific suitable constructs of the invention derived from RSV Fusion (F) protein, wherein Column K provides suitable sequences for F_DSCav1_mut0, Column L provides suitable sequences for F-del_DSCav1_mut0, Column M provides suitable sequences for F_DSCav1_mut4, Column N provides suitable sequences for F-del_DSCav1_mut4, Column O provides suitable sequences for F_DSCav1_mut5, Column P provides suitable sequences for F-del_DSCav1_mut5, Column Q provides suitable sequences for F_DSCav1_mut6, Column R provides suitable sequences for F-del_DSCav1_mut6, Column S provides suitable sequences for F_DSCav1_mut7, Column T provides suitable sequences for F-del_DSCav1_mut7, Column U provides suitable sequences for F_DSCav1_mut8, Column V provides suitable sequences for F-del_DSCav1_mut8.

The protein designs are indicated in the first row (Columns K to V), the specific protein SEQ ID NOs as provided in the sequence listing are in row 2, “PRT”. The SEQ ID NOs of corresponding coding sequences for each protein construct are provided in row 3, “cds” (compare with Table 4 for different cds optimizations). Further information e.g. regarding the type of codon modified coding sequence (opt1, opt2, opt3, opt4, opt5, opt6, opt11 etc.) is provided in the <223> identifier of the respective SEQ ID NO in the sequence listing and in Table 4. The SEQ ID NOs of corresponding mRNA constructs comprising said coding sequences and suitable 3′-UTRs and 5′-UTRs according to the invention are provided in each following row (mRNA designs “a-1” to “i-3” as specified herein).

In Table 6A, mRNA sequences derived from HRSV(A2) are provided, in Table 6B mRNA sequences derived from HRSV(Memphis-37) are provided

Further information e.g. regarding the type of coding sequence (wt, opt1, opt2, opt3, opt4, opt5, opt6, opt11 etc.) comprised in the mRNA constructs is provided in the <223> identifier of the respective SEQ ID NO in the sequence listing.

TABLE 6A Preferred mRNA constructs encoding RSV F (column K-V), derived from HRSV(A2) K L M N O P Q R S T U V PRT 3850 4219 4588 4957 5326 5695 6064 6433 6802 7171 7540 7909 cds 3851- 4220- 4589- 4958- 5327- 5696- 6065- 6434- 6803- 7172- 7541- 7910- 3858, 4227, 4596, 4965, 5334, 5703, 6072, 6441, 6810, 7179, 7548, 7917, 21367 21368 21375 21376 21377 21378 21379 21380 21381 21382 21383 21384 a-1 3859- 4228- 4597- 4966- 5335- 5704- 6073- 6442- 6811- 7180- 7549- 7918- 3866, 4235, 4604, 4973, 5342, 5711, 6080, 6449, 6818, 7187, 7556, 7925, 21427- 21430- 21451- 21454- 21457- 21460- 21463- 21466- 21469- 21472- 21475- 21478- 21429, 21432, 21453, 21456, 21459, 21462, 21465, 21468, 21471, 21474, 21477, 21480, 21573- 21576- 21597- 21600- 21603- 21606- 21609- 21612- 21615- 21618- 21621- 21624- 21575 21578 21599 21602 21605 21608 21611 21614 21617 21620 21623 21626 a-2 3867- 4236- 4605- 4974- 5343- 5712- 6081- 6450- 6819- 7188- 7557- 7926- 3874 4243 4612 4981 5350 5719 6088 6457 6826 7195 7564 7933 a-3 3875- 4244- 4613- 4982- 5351- 5720- 6089- 6458- 6827- 7196- 7565- 7934- 3882 4251 4620 4989 5358 5727 6096 6465 6834 7203 7572 7941 a-4 3883- 4252- 4621- 4990- 5359- 5728- 6097- 6466- 6835- 7204- 7573- 7942- 3890 4259 4628 4997 5366 5735 6104 6473 6842 7211 7580 7949 a-5 3891- 4260- 4629- 4998- 5367- 5736- 6105- 6474- 6843- 7212- 7581- 7950- 3898 4267 4636 5005 5374 5743 6112 6481 6850 7219 7588 7957 b-1 3899- 4268- 4637- 5006- 5375- 5744- 6113- 6482- 6851- 7220- 7589- 7958- 3906 4275 4644 5013 5382 5751 6120 6489 6858 7227 7596 7965 b-2 3907- 4276- 4645- 5014- 5383- 5752- 6121- 6490- 6859- 7228- 7597- 7966- 3914 4283 4652 5021 5390 5759 6128 6497 6866 7235 7604 7973 b-3 3915- 4284- 4653- 5022- 5391- 5760- 6129- 6498- 6867- 7236- 7605- 7974- 3922 4291 4660 5029 5398 5767 6136 6505 6874 7243 7612 7981 b-4 3923- 4292- 4661- 5030- 5399- 5768- 6137- 6506- 6875- 7244- 7613- 7982- 3930 4299 4668 5037 5406 5775 6144 6513 6882 7251 7620 7989 b-5 3931- 4300- 4669- 5038- 5407- 5776- 6145- 6514- 6883- 7252- 7621- 7990- 3938 4307 4676 5045 5414 5783 6152 6521 6890 7259 7628 7997 c-1 3939- 4308- 4677- 5046- 5415- 5784- 6153- 6522- 6891- 7260- 7629- 7998- 3946 4315 4684 5053 5422 5791 6160 6529 6898 7267 7636 8005 c-2 3947- 4316- 4685- 5054- 5423- 5792- 6161- 6530- 6899- 7268- 7637- 8006- 3954 4323 4692 5061 5430 5799 6168 6537 6906 7275 7644 8013 c-3 3955- 4324- 4693- 5062- 5431- 5800- 6169- 6538- 6907- 7276- 7645- 8014- 3962 4331 4700 5069 5438 5807 6176 6545 6914 7283 7652 8021 c-4 3963- 4332- 4701- 5070- 5439- 5808- 6177- 6546- 6915- 7284- 7653- 8022- 3970 4339 4708 5077 5446 5815 6184 6553 6922 7291 7660 8029 c-5 3971- 4340- 4709- 5078- 5447- 5816- 6185- 6554- 6923- 7292- 7661- 8030- 3978 4347 4716 5085 5454 5823 6192 6561 6930 7299 7668 8037 d-1 3979- 4348- 4717- 5086- 5455- 5824- 6193- 6562- 6931- 7300- 7669- 8038- 3986 4355 4724 5093 5462 5831 6200 6569 6938 7307 7676 8045 d-2 3987- 4356- 4725- 5094- 5463- 5832- 6201- 6570- 6939- 7308- 7677- 8046- 3994 4363 4732 5101 5470 5839 6208 6577 6946 7315 7684 8053 d-3 3995- 4364- 4733- 5102- 5471- 5840- 6209- 6578- 6947- 7316- 7685- 8054- 4002 4371 4740 5109 5478 5847 6216 6585 6954 7323 7692 8061 d-4 4003- 4372- 4741- 5110- 5479- 5848- 6217- 6586- 6955- 7324- 7693- 8062- 4010 4379 4748 5117 5486 5855 6224 6593 6962 7331 7700 8069 d-5 4011- 4380- 4749- 5118- 5487- 5856- 6225- 6594- 6963- 7332- 7701- 8070- 4018 4387 4756 5125 5494 5863 6232 6601 6970 7339 7708 8077 e-1 4019- 4388- 4757- 5126- 5495- 5864- 6233- 6602- 6971- 7340- 7709- 8078- 4026 4395 4764 5133 5502 5871 6240 6609 6978 7347 7716 8085 e-2 4027- 4396- 4765- 5134- 5503- 5872- 6241- 6610- 6979- 7348- 7717- 8086- 4034 4403 4772 5141 5510 5879 6248 6617 6986 7355 7724 8093 e-3 4035- 4404- 4773- 5142- 5511- 5880- 6249- 6618- 6987- 7356- 7725- 8094- 4042 4411 4780 5149 5518 5887 6256 6625 6994 7363 7732 8101 e-4 4043- 4412- 4781- 5150- 5519- 5888- 6257- 6626- 6995- 7364- 7733- 8102- 4050 4419 4788 5157 5526 5895 6264 6633 7002 7371 7740 8109 e-5 4051- 4420- 4789- 5158- 5527- 5896- 6265- 6634- 7003- 7372- 7741- 8110- 4058 4427 4796 5165 5534 5903 6272 6641 7010 7379 7748 8117 e-6 4059- 4428- 4797- 5166- 5535- 5904- 6273- 6642- 7011- 7380- 7749- 8118- 4066 4435 4804 5173 5542 5911 6280 6649 7018 7387 7756 8125 f-1 4067- 4436- 4805- 5174- 5543- 5912- 6281- 6650- 7019- 7388- 7757- 8126- 4074 4443 4812 5181 5550 5919 6288 6657 7026 7395 7764 8133 f-2 4075- 4444- 4813- 5182- 5551- 5920- 6289- 6658- 7027- 7396- 7765- 8134- 4082 4451 4820 5189 5558 5927 6296 6665 7034 7403 7772 8141 f-3 4083- 4452- 4821- 5190- 5559- 5928- 6297- 6666- 7035- 7404- 7773- 8142- 4090 4459 4828 5197 5566 5935 6304 6673 7042 7411 7780 8149 f-4 4091- 4460- 4829- 5198- 5567- 5936- 6305- 6674- 7043- 7412- 7781- 8150- 4098 4467 4836 5205 5574 5943 6312 6681 7050 7419 7788 8157 f-5 4099- 4468- 4837- 5206- 5575- 5944- 6313- 6682- 7051- 7420- 7789- 8158- 4106 4475 4844 5213 5582 5951 6320 6689 7058 7427 7796 8165 g-1 4107- 4476- 4845- 5214- 5583- 5952- 6321- 6690- 7059- 7428- 7797- 8166- 4114 4483 4852 5221 5590 5959 6328 6697 7066 7435 7804 8173 g-2 4115- 4484- 4853- 5222- 5591- 5960- 6329- 6698- 7067- 7436- 7805- 8174- 4122 4491 4860 5229 5598 5967 6336 6705 7074 7443 7812 8181 g-3 4123- 4492- 4861- 5230- 5599- 5968- 6337- 6706- 7075- 7444- 7813- 8182- 4130 4499 4868 5237 5606 5975 6344 6713 7082 7451 7820 8189 g-4 4131- 4500- 4869- 5238- 5607- 5976- 6345- 6714- 7083- 7452- 7821- 8190- 4138 4507 4876 5245 5614 5983 6352 6721 7090 7459 7828 8197 g-5 4139- 4508- 4877- 5246- 5615- 5984- 6353- 6722- 7091- 7460- 7829- 8198- 4146 4515 4884 5253 5622 5991 6360 6729 7098 7467 7836 8205 h-1 4147- 4516- 4885- 5254- 5623- 5992- 6361- 6730- 7099- 7468- 7837- 8206- 4154 4523 4892 5261 5630 5999 6368 6737 7106 7475 7844 8213 h-2 4155- 4524- 4893- 5262- 5631- 6000- 6369- 6738- 7107- 7476- 7845- 8214- 4162 4531 4900 5269 5638 6007 6376 6745 7114 7483 7852 8221 h-3 4163- 4532- 4901- 5270- 5639- 6008- 6377- 6746- 7115- 7484- 7853- 8222- 4170 4539 4908 5277 5646 6015 6384 6753 7122 7491 7860 8229 h-4 4171- 4540- 4909- 5278- 5647- 6016- 6385- 6754- 7123- 7492- 7861- 8230- 4178 4547 4916 5285 5654 6023 6392 6761 7130 7499 7868 8237 h-5 4179- 4548- 4917- 5286- 5655- 6024- 6393- 6762- 7131- 7500- 7869- 8238- 4186 4555 4924 5293 5662 6031 6400 6769 7138 7507 7876 8245 i-1 4187- 4556- 4925- 5294- 5663- 6032- 6401- 6770- 7139- 7508- 7877- 8246- 4194 4563 4932 5301 5670 6039 6408 6777 7146 7515 7884 8253 i-2 4195- 4564- 4933- 5302- 5671- 6040- 6409- 6778- 7147- 7516- 7885- 8254- 4210 4579 4948 5317 5686 6055 6424 6793 7162 7531 7900 8269 i-3 4211- 4580- 4949- 5318- 5687- 6056- 6425- 6794- 7163- 7532- 7901- 8270- 4218 4587 4956 5325 5694 6063 6432 6801 7170 7539 7908 8277

TABLE 6B Preferred mRNA constructs encoding RSV F (column K-V), derived from HRSV(Memphis-37) K L M N O P Q R S T U V PRT 13202 13571 16154 16523 16892 17261 17630 17999 18368 18737 19106 19475 cds 13203- 13572- 16155- 16524- 16893- 17262- 17631- 18000- 18369- 18738- 19107- 19476- 13210, 13579, 16162, 16531, 16900, 17269, 17638, 18007, 18376, 18745, 19114, 19483, 21393 21394 21401 21402 21403 21404 21405 21406 21407 21408 21409 21410 a-1 13211- 13580- 16163- 16532- 16901- 17270- 17639- 18008- 18377- 18746- 19115- 19484- 13218, 13587, 16170, 16539, 16908, 17277, 17646, 18015, 18384, 18753, 19122, 19491, 21499- 21502- 21523- 21526- 21529- 21532- 21535- 21538- 21541- 21544- 21547- 21550- 21501, 21504, 21525, 21528, 21531, 21534, 21537, 21540, 21543, 21546, 21549, 21552, 21645- 21648- 21669- 21672- 21675- 21678- 21681- 21684- 21687- 21690- 21693- 21696- 21647 21650 21671 21674 21677 21680 21683 21686 21689 21692 21695 21698 a-2 13219- 13588- 16171- 16540- 16909- 17278- 17647- 18016- 18385- 18754- 19123- 19492- 13226 13595 16178 16547 16916 17285 17654 18023 18392 18761 19130 19499 a-3 13227- 13596- 16179- 16548- 16917- 17286- 17655- 18024- 18393- 18762- 19131- 19500- 13234 13603 16186 16555 16924 17293 17662 18031 18400 18769 19138 19507 a-4 13235- 13604- 16187- 16556- 16925- 17294- 17663- 18032- 18401- 18770- 19139- 19508- 13242 13611 16194 16563 16932 17301 17670 18039 18408 18777 19146 19515 a-5 13243- 13612- 16195- 16564- 16933- 17302- 17671- 18040- 18409- 18778- 19147- 19516- 13250 13619 16202 16571 16940 17309 17678 18047 18416 18785 19154 19523 b-1 13251- 13620- 16203- 16572- 16941- 17310- 17679- 18048- 18417- 18786- 19155- 19524- 13258 13627 16210 16579 16948 17317 17686 18055 18424 18793 19162 19531 b-2 13259- 13628- 16211- 16580- 16949- 17318- 17687- 18056- 18425- 18794- 19163- 19532- 13266 13635 16218 16587 16956 17325 17694 18063 18432 18801 19170 19539 b-3 13267- 13636- 16219- 16588- 16957- 17326- 17695- 18064- 18433- 18802- 19171- 19540- 13274 13643 16226 16595 16964 17333 17702 18071 18440 18809 19178 19547 b-4 13275- 13644- 16227- 16596- 16965- 17334- 17703- 18072- 18441- 18810- 19179- 19548- 13282 13651 16234 16603 16972 17341 17710 18079 18448 18817 19186 19555 b-5 13283- 13652- 16235- 16604- 16973- 17342- 17711- 18080- 18449- 18818- 19187- 19556- 13290 13659 16242 16611 16980 17349 17718 18087 18456 18825 19194 19563 c-1 13291- 13660- 16243- 16612- 16981- 17350- 17719- 18088- 18457- 18826- 19195- 19564- 13298 13667 16250 16619 16988 17357 17726 18095 18464 18833 19202 19571 c-2 13299- 13668- 16251- 16620- 16989- 17358- 17727- 18096- 18465- 18834- 19203- 19572- 13306 13675 16258 16627 16996 17365 17734 18103 18472 18841 19210 19579 c-3 13307- 13676- 16259- 16628- 16997- 17366- 17735- 18104- 18473- 18842- 19211- 19580- 13314 13683 16266 16635 17004 17373 17742 18111 18480 18849 19218 19587 c-4 13315- 13684- 16267- 16636- 17005- 17374- 17743- 18112- 18481- 18850- 19219- 19588- 13322 13691 16274 16643 17012 17381 17750 18119 18488 18857 19226 19595 c-5 13323- 13692- 16275- 16644- 17013- 17382- 17751- 18120- 18489- 18858- 19227- 19596- 13330 13699 16282 16651 17020 17389 17758 18127 18496 18865 19234 19603 d-1 13331- 13700- 16283- 16652- 17021- 17390- 17759- 18128- 18497- 18866- 19235- 19604- 13338 13707 16290 16659 17028 17397 17766 18135 18504 18873 19242 19611 d-2 13339- 13708- 16291- 16660- 17029- 17398- 17767- 18136- 18505- 18874- 19243- 19612- 13346 13715 16298 16667 17036 17405 17774 18143 18512 18881 19250 19619 d-3 13347- 13716- 16299- 16668- 17037- 17406- 17775- 18144- 18513- 18882- 19251- 19620- 13354 13723 16306 16675 17044 17413 17782 18151 18520 18889 19258 19627 d-4 13355- 13724- 16307- 16676- 17045- 17414- 17783- 18152- 18521- 18890- 19259- 19628- 13362 13731 16314 16683 17052 17421 17790 18159 18528 18897 19266 19635 d-5 13363- 13732- 16315- 16684- 17053- 17422- 17791- 18160- 18529- 18898- 19267- 19636- 13370 13739 16322 16691 17060 17429 17798 18167 18536 18905 19274 19643 e-1 13371- 13740- 16323- 16692- 17061- 17430- 17799- 18168- 18537- 18906- 19275- 19644- 13378 13747 16330 16699 17068 17437 17806 18175 18544 18913 19282 19651 e-2 13379- 13748- 16331- 16700- 17069- 17438- 17807- 18176- 18545- 18914- 19283- 19652- 13386 13755 16338 16707 17076 17445 17814 18183 18552 18921 19290 19659 e-3 13387- 13756- 16339- 16708- 17077- 17446- 17815- 18184- 18553- 18922- 19291- 19660- 13394 13763 16346 16715 17084 17453 17822 18191 18560 18929 19298 19667 e-4 13395- 13764- 16347- 16716- 17085- 17454- 17823- 18192- 18561- 18930- 19299- 19668- 13402 13771 16354 16723 17092 17461 17830 18199 18568 18937 19306 19675 e-5 13403- 13772- 16355- 16724- 17093- 17462- 17831- 18200- 18569- 18938- 19307- 19676- 13410 13779 16362 16731 17100 17469 17838 18207 18576 18945 19314 19683 e-6 13411- 13780- 16363- 16732- 17101- 17470- 17839- 18208- 18577- 18946- 19315- 19684- 13418 13787 16370 16739 17108 17477 17846 18215 18584 18953 19322 19691 f-1 13419- 13788- 16371- 16740- 17109- 17478- 17847- 18216- 18585- 18954- 19323- 19692- 13426 13795 16378 16747 17116 17485 17854 18223 18592 18961 19330 19699 f-2 13427- 13796- 16379- 16748- 17117- 17486- 17855- 18224- 18593- 18962- 19331- 19700- 13434 13803 16386 16755 17124 17493 17862 18231 18600 18969 19338 19707 f-3 13435- 13804- 16387- 16756- 17125- 17494- 17863- 18232- 18601- 18970- 19339- 19708- 13442 13811 16394 16763 17132 17501 17870 18239 18608 18977 19346 19715 f-4 13443- 13812- 16395- 16764- 17133- 17502- 17871- 18240- 18609- 18978- 19347- 19716- 13450 13819 16402 16771 17140 17509 17878 18247 18616 18985 19354 19723 f-5 13451- 13820- 16403- 16772- 17141- 17510- 17879- 18248- 18617- 18986- 19355- 19724- 13458 13827 16410 16779 17148 17517 17886 18255 18624 18993 19362 19731 g-1 13459- 13828- 16411- 16780- 17149- 17518- 17887- 18256- 18625- 18994- 19363- 19732- 13466 13835 16418 16787 17156 17525 17894 18263 18632 19001 19370 19739 g-2 13467- 13836- 16419- 16788- 17157- 17526- 17895- 18264- 18633- 19002- 19371- 19740- 13474 13843 16426 16795 17164 17533 17902 18271 18640 19009 19378 19747 g-3 13475- 13844- 16427- 16796- 17165- 17534- 17903- 18272- 18641- 19010- 19379- 19748- 13482 13851 16434 16803 17172 17541 17910 18279 18648 19017 19386 19755 g-4 13483- 13852- 16435- 16804- 17173- 17542- 17911- 18280- 18649- 19018- 19387- 19756- 13490 13859 16442 16811 17180 17549 17918 18287 18656 19025 19394 19763 g-5 13491- 13860- 16443- 16812- 17181- 17550- 17919- 18288- 18657- 19026- 19395- 19764- 13498 13867 16450 16819 17188 17557 17926 18295 18664 19033 19402 19771 h-1 13499- 13868- 16451- 16820- 17189- 17558- 17927- 18296- 18665- 19034- 19403- 19772- 13506 13875 16458 16827 17196 17565 17934 18303 18672 19041 19410 19779 h-2 13507- 13876- 16459- 16828- 17197- 17566- 17935- 18304- 18673- 19042- 19411- 19780- 13514 13883 16466 16835 17204 17573 17942 18311 18680 19049 19418 19787 h-3 13515- 13884- 16467- 16836- 17205- 17574- 17943- 18312- 18681- 19050- 19419- 19788- 13522 13891 16474 16843 17212 17581 17950 18319 18688 19057 19426 19795 h-4 13523- 13892- 16475- 16844- 17213- 17582- 17951- 18320- 18689- 19058- 19427- 19796- 13530 13899 16482 16851 17220 17589 17958 18327 18696 19065 19434 19803 h-5 13531- 13900- 16483- 16852- 17221- 17590- 17959- 18328- 18697- 19066- 19435- 19804- 13538 13907 16490 16859 17228 17597 17966 18335 18704 19073 19442 19811 i-1 13539- 13908- 16491- 16860- 17229- 17598- 17967- 18336- 18705- 19074- 19443- 19812- 13546 13915 16498 16867 17236 17605 17974 18343 18712 19081 19450 19819 i-2 13547- 13916- 16499- 16868- 17237- 17606- 17975- 18344- 18713- 19082- 19451- 19820- 13562 13931 16514 16883 17252 17621 17990 18359 18728 19097 19466 19835 i-3 13563- 13932- 16515- 16884- 17253- 17622- 17991- 18360- 18729- 19098- 19467- 19836- 13570 13939 16522 16891 17260 17629 17998 18367 18736 19105 19474 19843

In preferred embodiments, the artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of Seq ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 21415-21480, 21561-21626, 11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843, 21489-21552, 21635-21698 or a fragment or variant of any of these sequences, wherein, optionally, at least one or more than one, or wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.

In preferred embodiments, the artificial RNA comprises (a) at least one heterologous 5′ untranslated region (5′-UTR) and/or at least one heterologous 3′ untranslated region (3′-UTR) and (b) at least one coding sequence operably linked to said 3′-UTR and/or 5′-UTR encoding at least one antigenic peptide or protein derived from a RSV F or a fragment or variant thereof, wherein

-   -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 78-482, 11735-12094, 21415-21417,         21561-21563, 21489, 21490, 21635, 21636 (encoding F0) or a         fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 493-897, 12104-12463, 21418-21420,         21564-21566, 21491, 21492, 21637, 21638 (encoding F-del) or a         fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 907-1266, 12473-12832, 21421-21423,         21567-21569, 21493-21495, 21639-21641 (encoding F0_DSCav1) or a         fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 1276-1635, 8278, 12842-13201,         21424-21426, 21570-21572, 21496-21498, 21642-21644 (encoding         F-del_DSCav1) or a fragment or variant of any of these         sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 1645-2004, 13949-14308, 21433-21435,         21579-21581, 21505-21507, 21651-21653 (encoding F_DSCav1_mut1)         or a fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 2014-2373, 14318-14677, 21436-21438,         21582-21584, 21508-21510, 21654-21656 (encoding         F-del_DSCav1_mut1) or a fragment or variant of any of these         sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 2383-2742, 14687-15046, 21439-21441,         21585-21587, 21511-21513, 21657-21659 (encoding F_DSCav1_mut2)         or a fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 2752-3111, 15056-15415, 21442-21444,         21588-21590, 21514-21516, 21660-21662 (encoding         F-del_DSCav1_mut2) or a fragment or variant of any of these         sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 3121-3480, 15425-15784, 21445-21447,         21591-21593, 21517-21519, 21663-21665 (encoding F_DSCav1_mut3)         or a fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 3490-3849, 15794-16153, 21448-21450,         21594-21596, 21520-21522, 21666-21668 (encoding         F-del_DSCav1_mut3) or a fragment or variant of any of these         sequences.     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 3859-4218, 13211-13570, 21427-21429,         21573-21575, 21499-21501, 21645-21647 (encoding F_DSCav1_mut0)         or a fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 4228-4587, 13580-13939, 21430-21432,         21576-21578, 21502-21504, 21648-21650 (encoding         F-del_DSCav1_mut0) or a fragment or variant of any of these         sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 4597-4956, 16163-16522, 21451-21453,         21597-21599, 21523-21525, 21669-21671 (encoding F_DSCav1_mut4)         or a fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 4966-5325, 16532-16891, 21454-21456,         21600-21602, 21526-21528, 21672-21674 (encoding         F-del_DSCav1_mut4) or a fragment or variant of any of these         sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 5335-5694, 16901-17260, 21457-21459,         21603-21605, 21529-21531, 21675-21677 (encoding F_DSCav1_mut5)         or a fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 5704-6063, 17270-17629, 21460-21462,         21606-21608, 21532-21534, 21678-21680 (encoding         F-del_DSCav1_mut5) or a fragment or variant of any of these         sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 6073-6432, 17639-17998, 21463-21465,         21609-21611, 21535-21537, 21681-21683 (encoding F_DSCav1_mut6)         or a fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 6442-6801, 18008-18367, 21466-21468,         21612-21614, 21538-21540, 21684-21686 (encoding         F-del_DSCav1_mut6) or a fragment or variant of any of these         sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 6811-7170, 18377-18736, 21469-21471,         21615-21617, 21541-21543, 21687-21689 (encoding F_DSCav1_mut7)         or a fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 7180-7539, 18746-19105, 21472-21474,         21618-21620, 21544-21546, 21690-21692 (encoding         F-del_DSCav1_mut7) or a fragment or variant of any of these         sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 7549-7908, 19115-19474, 21475-21477,         21621-21623, 21547-21549, 21693-21695 (encoding F_DSCav1_mut8)         or a fragment or variant of any of these sequences;     -   said artificial RNA comprises or consists of an RNA sequence         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 7918-8277, 19484-19843, 21478-21480,         21624-21626, 21550-21552, 21696-21698 (encoding         F-del_DSCav1_mut8) or a fragment or variant of any of these         sequences, and         wherein, optionally, at least one or more than one, or wherein         all uracil nucleotides are replaced by pseudouridine (ψ)         nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.

In particularly preferred embodiments, the artificial RNA comprises (a) at least one heterologous 5′ untranslated region (5′-UTR) and/or at least one heterologous 3′ untranslated region (3′-UTR) and (b) at least one coding sequence operably linked to said 3′-UTR and/or 5′-UTR encoding at least one antigenic peptide or protein derived from a RSV F or a fragment or variant thereof, wherein said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5704-6063, 17270-17629, 21460-21462, 21606-21608, 21532-21534, 21678-21680 (encoding F-del_DSCav1_mut5) or a fragment or variant of any of these sequences.

As outlined throughout the specification, additional information regarding suitable amino acid sequences or nucleic acid sequences (coding sequences, mRNA sequences) may also be derived from the sequence listing, in particular from the details provided therein under identifier <223> as explained in the following.

For example, the numeric identifier <223> in the sequence listing of SEQ ID NO: 68 reads as follows: “derived and/or modified protein sequence (artificial) from HRSV(A2)_F0”. It has to be noted that throughout the sequence listing, information provided under numeric identifier <223> follows the same structure: “<SEQUENCE_DESCRIPTOR> from <CONSTRUCT_IDENTIFIER>”. The <SEQUENCE_DESCRIPTOR> relates to the type of sequence (e.g., “derived and/or modified protein sequence”, “derived and/or modified CDS” “mRNA product Design a-1 comprising derived and/or modified sequence”, or “mRNA product Design b-4 comprising derived and/or modified sequence”, or “mRNA product Design c-5 comprising derived and/or modified sequence”, or “mRNA product Design g-4 comprising derived and/or modified sequence” etc.) and whether the sequence comprises or consists of a wild type sequence (“wt”) or whether the sequence comprises or consists of a sequence-optimized sequence (e.g. “opt1”, “opt2”, “opt3”, “opt4”, “opt5”, “opt6”, “opt11”; sequence optimizations are described in further detail below). For example, the <SEQUENCE_DESCRIPTOR> provided under numeric identifier <223> of SEQ ID NO: 68 reads as follows: “derived and/or modified protein sequence (artificial)”. The <CONSTRUCT_IDENTIFIER> provided under numeric identifier <223> has the following structures: (“organism_construct name”, or “organism__accession number__construct name”) and is intended to help the person skilled in the art to explicitly derive suitable nucleic acid sequences (e.g., RNA, mRNA) encoding the same RSV protein according to the invention. For example, the <CONSTRUCT_IDENTIFIER> provided under numeric identifier <223> of SEQ ID NO: 68 reads as follows: “HRSV(A2)_F0”. In that example, the respective protein sequence is derived from “HRSV(A2)” (organism), wherein the protein comprises the structural elements “F0” (construct name, full-length F). If the skilled person uses the construct identifier of SEQ ID NO: 68, namely “HRSV(A2)_F0”, said person easily arrives at a list of suitable nucleic acid coding sequences, e.g. RNA coding sequences and mRNA sequences that can easily be retrieve from the sequence listing of the present invention.

Composition:

A second aspect relates to a composition comprising at least one artificial RNA of the first aspect.

In a preferred embodiment of the second aspect, the composition comprises at least one artificial RNA of the first aspect and, optionally, at least one pharmaceutically acceptable carrier.

The term “pharmaceutically acceptable carrier” as used herein preferably includes the liquid or non-liquid basis of the composition. If the composition is provided in liquid form, the carrier will preferably be water, typically pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions. Water or preferably a buffer, more preferably an aqueous buffer, may be used, containing a sodium salt, preferably at least 50 mM of a sodium salt, a calcium salt, preferably at least 0.01 mM of a calcium salt, and optionally a potassium salt, preferably at least 3 mM of a potassium salt. According to a preferred embodiment, the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Without being limited thereto, examples of sodium salts include e.g. NaCl, NaI, NaBr, Na₂CO₃, NaHC₃, Na₂SO₄, examples of the optional potassium salts include e.g. KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, and examples of calcium salts include e.g. CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂. Furthermore, organic anions of the aforementioned cations may be contained in the buffer.

In embodiments, the composition as defined herein may comprise a plurality or at least more than one of the artificial RNAs as defined in the context of the first aspect or the second aspect of the invention.

In embodiments, the at least one RNA comprised in the composition is a bi- or multicistronic nucleic acid, particularly a bi- or multicistronic nucleic acid as defined herein, which encodes the at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve distinct antigenic peptides or protein derived from the same RSV and/or a different RSV.

In embodiment, the composition may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different artificial RNAs as defined in the context of the first aspect of the invention each encoding at least one antigenic peptide or protein derived from genetically the same RSV or a fragment or variant thereof. The terms “same” or “same RSV” as used in the context of a virus, e.g. “same virus”, have to be understood as genetically the same. Particularly, said (genetically) same virus expresses the same proteins or peptides, wherein all proteins or peptides have the same amino acid sequence. Particularly, said (genetically) same RSV expresses essentially the same proteins, peptides or polyproteins, wherein these protein, peptide or polyproteins preferably do not differ in their amino acid sequence(s). A non-limiting list of exemplary RSV viruses is provided in List 1.

In embodiments, the composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different RNAs as defined in the context of the first aspect of the invention each encoding at least one peptide or protein derived from a genetically different RSV or a fragment or variant thereof. The terms “different” or “different RSV” as used throughout the present specification in the context of a virus, e.g. “different” virus, has to be understood as the difference between at least two respective viruses, wherein the difference is manifested on the RNA genome of the respective different virus. Particularly, said (genetically) different RSV expresses at least one different protein, peptide or polyprotein, wherein the at least one different protein, peptide or polyprotein preferably differs in at least one amino acid.

In other embodiments, the composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more further RNA constructs encoding RSV antigens selected from glycoprotein G, short hydrophobic protein SH, matrix protein M, nucleoprotein N, large polymerase L, M2-1 protein, M2-2 protein, phosphoprotein P, non-structural protein NS1 or non-structural protein NS2, or any combination thereof.

For the production of a composition comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 further RNA constructs encoding RSV, methods as disclosed in the PCT application PCT/EP2016/082487 or in published patent application WO2017/1090134A1 are preferably used and adapted accordingly.

In preferred embodiments, the composition of the second aspect comprises at least one artificial RNA of the first aspect and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV selected from matrix protein M, nucleoprotein N, M2-1 protein, and/or phosphoprotein P or combinations thereof. The addition of a further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M, N, M2-1, and/or phosphoprotein P (or combinations thereof) is particularly advantageous to e.g. promote a T-cell immune response.

Notably, embodiments relating to the artificial RNA of the first aspect may likewise be read on and be understood as suitable embodiments of the at least one further artificial RNA of the second aspect (e.g., embodiments relating to UTR combinations, cds optimizations, histone stem loop, PolyA, PolyC, cap structure, mRNA structure, mRNA production and mRNA purification etc.).

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV matrix protein M.

Alternatively, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV N.

Alternatively, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M2-1.

Alternatively, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV P.

In preferred embodiments, the composition of the second aspect comprises two further artificial RNA species each comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV selected from matrix protein M, nucleoprotein N, M2-1, and phosphoprotein P.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV N.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV N, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M2-1.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV P, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M2-1.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV P, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV N.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M2-1.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV P.

In preferred embodiments, the composition of the second aspect comprises three further artificial RNA species each comprising at comprising three further artificial RNA species each comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV selected from matrix protein M, nucleoprotein N, M2-1, and phosphoprotein P.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV N, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV P.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M2-1, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV N, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV P.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M2-1, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV P.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M2-1, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV N.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M2-1.

In preferred embodiments, the composition of the second aspect comprises four further artificial RNA species each comprising at comprising three further artificial RNA species each comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV selected from matrix protein M, nucleoprotein N, M2-1, and phosphoprotein P.

Accordingly, the composition of the second aspect may suitably comprise at least one artificial RNA of the first aspect encoding RSF F, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV N, at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV P, and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV M2-1.

In preferred embodiments of the second aspect, the coding sequence of the further artificial RNA encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 9684, 10053-10133, 10134, 10503-10636, 10637, 11006-11182, 11183, 11552-11725, 19844, 20213, 20582, 20951 or a fragment or variant of any of these sequences. Additional information regarding each of these suitable amino acid sequences encoding RSV proteins may also be derived from the sequence listing, in particular from the details provided therein under identifier <223> as explained in the following.

In further embodiments of the second aspect, the coding sequence of the further artificial RNA encodes at least one antigenic peptide or protein as defined herein and additionally a heterologous secretory signal sequence or heterologous secretory signal peptide. The heterologous secretory signal sequence may increase the secretion of the encoded antigenic peptide or protein.

Suitable secretory signal peptides may be selected from the list of amino acid sequences according to SEQ ID NOs: 1-1115 and SEQ ID NO: 1728 of the patent application WO2017/081082, or fragments or variants of these sequences. On nucleic acid level, any nucleic acid sequence (e.g. RNA sequence) may be selected which encodes such amino acid sequences. In this context, the disclosure of WO2017/081082 is herewith incorporated by reference.

According to embodiments, the secretory signal sequence comprises an amino acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 21329-21362 or a fragment or variant of any of these sequences. Additional information regarding each of these suitable amino acid sequences encoding secretory signal sequences may also be derived from the sequence listing, in particular from the details provided therein under identifier <223>.

In preferred embodiments, the further artificial RNA of the composition comprises

-   a) at least one heterologous 5′ untranslated region (5′-UTR) and/or     at least one heterologous 3′ untranslated region (3′-UTR); and -   b) at least one coding sequence operably linked to said 3′-UTR     and/or 5′-UTR encoding at least one antigenic peptide or protein     derived from a RSV M, N, M2-1, and/or phosphoprotein P or a fragment     or variant thereof, preferably encoding an amino acid sequence     selected from any one of SEQ ID NOs: 9684, 10053-10133, 10134,     10503-10636, 10637, 11006-11182, 11183, 11552-11725, 19844, 20213,     20582, 20951 or a fragment or variant of any of these sequences.

In preferred embodiments, the further artificial RNA of the composition comprises

-   a) at least one heterologous 5′ untranslated region (5′-UTR) and/or     at least one heterologous 3′ untranslated region (3′-UTR); and -   b) at least one coding sequence operably linked to said 3′-UTR     and/or 5′-UTR encoding at least one antigenic peptide or protein     derived from a RSV M2-1 or a fragment or variant thereof, preferably     encoding an amino acid sequence selected from any one of SEQ ID NOs:     11183, 20953, 21414 or a fragment or variant of any of these     sequences.

Suitably, the further artificial RNA of the composition comprises a coding sequence is operably linked to a 3′-UTR and a 5′-UTR selected from a-1, a-2, a-3, a-4, a-5, b-1, b-2, b-3, b-4, b-5, c-1, c-2, c-3, c-4, c-5, d-1, d-2, d-3, d-4, d-5, e-1, e-2, e-3, e-4, e-5, e-6, f-1, f-2, f-3, f-4, f-5, g-1, g-2, g-3, g-4, g-5, h-1, h-2, h- 3, h-4, h-5, i-1, i-2, or i-3 (as defined in the context of the first aspect).

Suitably, the further artificial RNA of the composition comprises a coding sequence wherein at least one or more than one, or wherein preferably all uracil nucleotides are replaced by pseudouridine (L) nucleotides or N1-methylpseudouridine (m1L) nucleotides.

Accordingly, in other embodiments, the further artificial RNA of of the composition may comprise a 5′-cap sequence element according to SEQ ID NOs: 43 or 21321, or a fragment or variant thereof.

In preferred embodiments of the second aspect, the coding sequence of the further RNA comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 9685-9692, 10135-10142, 10638-10645, 11184-11119, 19845-19852, 20214-20221, 20583-20590, 20952-20959, 21385-21388, 21411-21414 or a fragment or a fragment or variant of any of these sequences (cds optimizations as defined in the context of the first aspect). Additional information regarding each of these suitable amino acid sequences encoding RSV proteins may also be derived from the sequence listing, in particular from the details provided therein under identifier <223> as explained in the following.

In preferred embodiments of the second aspect, the further artificial RNA, preferably the further mRNA of the composition comprises preferably in 5′- to 3-direction the following elements a)-i):

-   a) 5′-cap structure, preferably as specified in the context of the     first aspect, most preferably a Cap1 structure; -   b) optionally, 5′-UTR as specified in the context of the first     aspect, preferably at least one selected from SEQ ID NOs: 1-22; -   c) a ribosome binding site, preferably as specified herein; d) at     least one coding sequence selected from SEQ ID NOs: 9685-9692,     10135-10142, 10638-10645, 11184-11119, 19845-19852, 20214-20221,     20583-20590, 20952-20959, 21385-21388, 21411-21414; -   e) 3′-UTR as as specified in the context of the first aspect,     preferably at least one selected from SEQ ID NOs: 23-38; -   f) optionally, a poly(A) sequence, preferably as specified in the     context of the first aspect; -   g) optionally, a poly(C) sequence, preferably as specified in the     context of the first aspect; -   h) optionally, a histone stem-loop, preferably as specified in the     context of the first aspect; -   i) optionally, a 3′-terminal sequence element as specified in the     context of the first aspect, preferably     -   according to according to SEQ ID NOs: 44-63, or 21322-21328, and         wherein optionally at least one or more than one, preferably         wherein all uracil nucleotides are replaced by pseudouridine (ψ)         nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.

In particularly preferred embodiments of the second aspect, the further artificial RNA, preferably the further mRNA of the composition comprises preferably in 5′- to 3′-direction the following elements a)-i):

-   a) 5′-cap structure, preferably as specified in the context of the     first aspect, most preferably a Cap1 structure; -   b) optionally, 5′-UTR as specified in the context of the first     aspect, preferably at least one selected from SEQ ID NOs: 1-22; -   c) a ribosome binding site, preferably as specified herein; -   d) at least one coding sequence selected from SEQ ID NOs:     11185-11191, 21388, 20952-20959, 21414; -   e) 3′-UTR as as specified in the context of the first aspect,     preferably at least one selected from SEQ ID NOs: 23-38; -   f) optionally, a poly(A) sequence, preferably as specified in the     context of the first aspect; -   g) optionally, a poly(C) sequence, preferably as specified in the     context of the first aspect; -   h) optionally, a histone stem-loop, preferably as specified in the     context of the first aspect; -   i) optionally, a 3′-terminal sequence element as specified in the     context of the first aspect, preferably according to according to     SEQ ID NOs: 44-63, or 21322-21328, and     -   wherein optionally at least one or more than one, preferably         wherein all uracil nucleotides are replaced by pseudouridine         (Ly) nucleotides or N1-methylpseudouridine (m1Ly) nucleotides.

In that context, preferred RSV polypeptide, nucleic acid, and mRNA sequences of the second aspect are provided in Table 7A.

In Table 7A, Columns A to D represent specific suitable constructs of the second aspect derived from RSV protein, wherein Column A provides suitable sequences for M, Column B provides suitable sequences for N, Column C provides suitable sequences for P, and Column D provides suitable sequences M2-1.

The protein names/designs are indicated in row 1 (Columns A to D), the protein SEQ ID NOs as provided in the sequence listing are in row 2 (“PRT”). Preferred protein SEQ ID NOs of HRSV(A2) are provided in row 3 (“HRSV(A2) PRT”). The SEQ ID NOs of corresponding coding sequences for each indicated HRSV(A2) protein are provided in rows 4-11 (“cds wt”, “cds opt1”, “cds opt2”, “cds opt3”, “cds opt4”, “cds opt5”, “cds opt6”, “cds opt11”, respectively). The SEQ ID NOs of corresponding mRNA constructs comprising said coding sequences and suitable 3′-UTRs and 5′-UTRs according to the invention are provided in rows 12 to 55 (“mRNA design a-1” to “mRNA design i-3” as specified herein).

Preferred protein SEQ ID NOs of HRSV(Memphis-37) are provided in row 56 (“Memphis-37 PRT”). The SEQ ID NOs of corresponding coding sequences for each indicated HRSV(Memphis-37) protein are provided in rows 57-64 (“cds wt”, “cds opt1”, “cds opt2”, “cds opt3”, “cds opt4”, “cds opt5”, “cds opt6”, “cds opt11”, respectively).

The SEQ ID NOs of corresponding mRNA constructs comprising said coding sequences and suitable 3′-UTRs and 5′-UTRs according to the invention are provided in rows 65 to 108 (“mRNA design a-” to “mRNA design i-3” as specified herein).

Further information e.g. regarding the type of coding sequence comprised in the indicated mRNA constructs is provided in the <223> identifier of the respective SEQ ID NO in the sequence listing.

TABLE 7A Preferred further coding sequences and mRNA constructs of the composition or vaccine A B C D 1 Strain Description RSV M RSV N RSV P RSV M2-1 2 divers PRT 10053-10133 10503-10636 11006-11182 11552-11725 3 HRSV(A2) PRT 9684 10134 10637 11183 4 HRSV(A2) cds wt 9685 10135 10638 11184 5 HRSV(A2) CDS opt1 9686, 21385 10136, 21386 10639, 21387 11185, 21388 6 HRSV(A2) cds opt2 9687 10137 10640 11186 7 HRSV(A2) cds opt3 9688 10138 10641 11187 8 HRSV(A2) cds opt4 9689 10139 10642 11188 9 HRSV(A2) cds opt5 9690 10140 10643 11189 10 HRSV(A2) cds opt6 9691 10141 10644 11190 11 HRSV(A2) cds opt11 9692 10142 10645 11191 12 HRSV(A2) mRNA Design a-1 9693-9700, 10143-10150, 10646-10653, 11192-11199, 21481, 21482, 21483, 21484, 21485, 21486, 21487, 21488, 21627, 21628 21629, 21630 21631, 21632 21633, 21634 13 HRSV(A2) mRNA Design a-2 9701-9708 10151-10158 10654-10661 11200-11207 14 HRSV(A2) mRNA Design a-3 9709-9716 10159-10166 10662-10669 11208-11215 15 HRSV(A2) mRNA Design a-4 9717-9724 10167-10174 10670-10677 11216-11223 16 HRSV(A2) mRNA Design a-5 9725-9732 10175-10182 10678-10685 11224-11231 17 HRSV(A2) mRNA Design b-1 9733-9740 10183-10190 10686-10693 11232-11239 18 HRSV(A2) mRNA Design b-2 9741-9748 10191-10198 10694-10701 11240-11247 19 HRSV(A2) mRNA Design b-3 9749-9756 10199-10206 10702-10709 11248-11255 20 HRSV(A2) mRNA Design b-4 9757-9764 10207-10214 10710-10717 11256-11263 21 HRSV(A2) mRNA Design b-5 9765-9772 10215-10222 10718-10725 11264-11271 22 HRSV(A2) mRNA Design c-1 9773-9780 10223-10230 10726-10733 11272-11279 23 HRSV(A2) mRNA Design c-2 9781-9788 10231-10238 10734-10741 11280-11287 24 HRSV(A2) mRNA Design c-3 9789-9796 10239-10246 10742-10749 11288-11295 25 HRSV(A2) mRNA Design c-4 9797-9804 10247-10254 10750-10757 11296-11303 26 HRSV(A2) mRNA Design c-5 9805-9812 10255-10262 10758-10765 11304-11311 27 HRSV(A2) mRNA Design d-1 9813-9820 10263-10270 10766-10773 11312-11319 28 HRSV(A2) mRNA Design d-2 9821-9828 10271-10278 10774-10781 11320-11327 29 HRSV(A2) mRNA Design d-3 9829-9836 10279-10286 10782-10789 11328-11335 30 HRSV(A2) mRNA Design d-4 9837-9844 10287-10294 10790-10797 11336-11343 31 HRSV(A2) mRNA Design d-5 9845-9852 10295-10302 10798-10805 11344-11351 32 HRSV(A2) mRNA Design e-1 9853-9860 10303-10310 10806-10813 11352-11359 33 HRSV(A2) mRNA Design e-2 9861-9868 10311-10318 10814-10821 11360-11367 34 HRSV(A2) mRNA Design e-3 9869-9876 10319-10326 10822-10829 11368-11375 35 HRSV(A2) mRNA Design e-4 9877-9884 10327-10334 10830-10837 11376-11383 36 HRSV(A2) mRNA Design e-5 9885-9892 10335-10342 10838-10845 11384-11391 37 HRSV(A2) mRNA Design e-6 9893-9900 10343-10350 10846-10853 11392-11399 38 HRSV(A2) mRNA Design f-1 9901-9908 10351-10358 10854-10861 11400-11407 39 HRSV(A2) mRNA Design f-2 9909-9916 10359-10366 10862-10869 11408-11415 40 HRSV(A2) mRNA Design f-3 9917-9924 10367-10374 10870-10877 11416-11423 41 HRSV(A2) mRNA Design f-4 9925-9932 10375-10382 10878-10885 11424-11431 42 HRSV(A2) mRNA Design f-5 9933-9940 10383-10390 10886-10893 11432-11439 43 HRSV(A2) mRNA Design g-1 9941-9948 10391-10398 10894-10901 11440-11447 44 HRSV(A2) mRNA Design g-2 9949-9956 10399-10406 10902-10909 11448-11455 45 HRSV(A2) mRNA Design g-3 9957-9964 10407-10414 10910-10917 11456-11463 46 HRSV(A2) mRNA Design g-4 9965-9972 10415-10422 10918-10925 11464-11471 47 HRSV(A2) mRNA Design g-5 9973-9980 10423-10430 10926-10933 11472-11479 48 HRSV(A2) mRNA Design h-1 9981-9988 10431-10438 10934-10941 11480-11487 49 HRSV(A2) mRNA Design h-2 9989-9996 10439-10446 10942-10949 11488-11495 50 HRSV(A2) mRNA Design h-3  9997-10004 10447-10454 10950-10957 11496-11503 51 HRSV(A2) mRNA Design h-4 10005-10012 10455-10462 10958-10965 11504-11511 52 HRSV(A2) mRNA Design h-5 10013-10020 10463-10470 10966-10973 11512-11519 53 HRSV(A2) mRNA Design i-1 10021-10028 10471-10478 10974-10981 11520-11527 54 HRSV(A2) mRNA Design i-2 10029-10044 10479-10494 10982-10997 11528-11543 55 HRSV(A2) mRNA Design i-3 10045-10052 10495-10502 10998-11005 11544-11551 56 Memphis-37 PRT 19844 20213 20582 20951 57 Memphis-37 CDS wt 19845 20214 20583 20952 58 Memphis-37 CDS opt1 19846, 21411 20215, 21412 20584, 21413 20953, 21414 59 Memphis-37 CDS opt2 19847 20216 20585 20954 60 Memphis-37 CDS opt3 19848 20217 20586 20955 61 Memphis-37 CDS opt4 19849 20218 20587 20956 62 Memphis-37 CDS opt5 19850 20219 20588 20957 63 Memphis-37 CDS opt6 19851 20220 20589 20958 64 Memphis-37 CDS opt11 19852 20221 20590 20959 65 Memphis-37 mRNA Design a-1 19853-19860, 20222-20229, 20591-20598, 20960-20967, 21553, 21554, 21555, 21556, 21557, 21558, 21559, 21560, 21699, 21700 21701, 21702 21703, 21704 21705, 21706 66 Memphis-37 mRNA Design a-2 19861-19868 20230-20237 20599-20606 20968-20975 67 Memphis-37 mRNA Design a-3 19869-19876 20238-20245 20607-20614 20976-20983 68 Memphis-37 mRNA Design a-4 19877-19884 20246-20253 20615-20622 20984-20991 69 Memphis-37 mRNA Design a-5 19885-19892 20254-20261 20623-20630 20992-20999 70 Memphis-37 mRNA Design b-1 19893-19900 20262-20269 20631-20638 21000-21007 71 Memphis-37 mRNA Design b-2 19901-19908 20270-20277 20639-20646 21008-21015 72 Memphis-37 mRNA Design b-3 19909-19916 20278-20285 20647-20654 21016-21023 73 Memphis-37 mRNA Design b-4 19917-19924 20286-20293 20655-20662 21024-21031 74 Memphis-37 mRNA Design b-5 19925-19932 20294-20301 20663-20670 21032-21039 75 Memphis-37 mRNA Design c-1 19933-19940 20302-20309 20671-20678 21040-21047 76 Memphis-37 mRNA Design c-2 19941-19948 20310-20317 20679-20686 21048-21055 77 Memphis-37 mRNA Design c-3 19949-19956 20318-20325 20687-20694 21056-21063 78 Memphis-37 mRNA Design c-4 19957-19964 20326-20333 20695-20702 21064-21071 79 Memphis-37 mRNA Design c-5 19965-19972 20334-20341 20703-20710 21072-21079 80 Memphis-37 mRNA Design d-1 19973-19980 20342-20349 20711-20718 21080-21087 81 Memphis-37 mRNA Design d-2 19981-19988 20350-20357 20719-20726 21088-21095 82 Memphis-37 mRNA Design d-3 19989-19996 20358-20365 20727-20734 21096-21103 83 Memphis-37 mRNA Design d-4 19997-20004 20366-20373 20735-20742 21104-21111 84 Memphis-37 mRNA Design d-5 20005-20012 20374-20381 20743-20750 21112-21119 85 Memphis-37 mRNA Design e-1 20013-20020 20382-20389 20751-20758 21120-21127 86 Memphis-37 mRNA Design e-2 20021-20028 20390-20397 20759-20766 21128-21135 87 Memphis-37 mRNA Design e-3 20029-20036 20398-20405 20767-20774 21136-21143 88 Memphis-37 mRNA Design e-4 20037-20044 20406-20413 20775-20782 21144-21151 89 Memphis-37 mRNA Design e-5 20045-20052 20414-20421 20783-20790 21152-21159 90 Memphis-37 mRNA Design e-6 20053-20060 20422-20429 20791-20798 21160-21167 91 Memphis-37 mRNA Design f-1 20061-20068 20430-20437 20799-20806 21168-21175 92 Memphis-37 mRNA Design f-2 20069-20076 20438-20445 20807-20814 21176-21183 93 Memphis-37 mRNA Design f-3 20077-20084 20446-20453 20815-20822 21184-21191 94 Memphis-37 mRNA Design f-4 20085-20092 20454-20461 20823-20830 21192-21199 95 Memphis-37 mRNA Design f-5 20093-20100 20462-20469 20831-20838 21200-21207 96 Memphis-37 mRNA Design g-1 20101-20108 20470-20477 20839-20846 21208-21215 97 Memphis-37 mRNA Design g-2 20109-20116 20478-20485 20847-20854 21216-21223 98 Memphis-37 mRNA Design g-3 20117-20124 20486-20493 20855-20862 21224-21231 99 Memphis-37 mRNA Design g-4 20125-20132 20494-20501 20863-20870 21232-21239 100 Memphis-37 mRNA Design g-5 20133-20140 20502-20509 20871-20878 21240-21247 101 Memphis-37 mRNA Design h-1 20141-20148 20510-20517 20879-20886 21248-21255 102 Memphis-37 mRNA Design h-2 20149-20156 20518-20525 20887-20894 21256-21263 103 Memphis-37 mRNA Design h-3 20157-20164 20526-20533 20895-20902 21264-21271 104 Memphis-37 mRNA Design h-4 20165-20172 20534-20541 20903-20910 21272-21279 105 Memphis-37 mRNA Design h-5 20173-20180 20542-20549 20911-20918 21280-21287 106 Memphis-37 mRNA Design i-1 20181-20188 20550-20557 20919-20926 21288-21295 107 Memphis-37 mRNA Design i-2 20189-20204 20558-20573 20927-20942 21296-21311 108 Memphis-37 mRNA Design i-3 20205-20212 20574-20581 20943-20950 21312-21319

Accordingly, in preferred embodiments, the composition of the second aspect comprises at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV selected from matrix protein M, nucleoprotein N, M2-1 protein, phosphoprotein P, wherein the further artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 9693-10052, 10143-10502, 10646-11005, 11192-11551, 19853-20212, 20222-20581, 20591-20950, 20960-21319, 21481-21488, 21627-21634, 21553-21560, 21699-21706 or a fragment or variant of any of these sequences, wherein, optionally, at least one or more than one, or wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides or N1-methylpseudouridine (m1 ψ) nucleotides. Additional information regarding each of these suitable amino acid sequences encoding RSV proteins may also be derived from the sequence listing, in particular from the details provided therein under identifier <223> as explained in the following.

In particularly preferred embodiments of the second aspect, the composition of the second aspect comprises

-   -   at least one artificial RNA which is identical or at least 70%,         80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,         97%, 98%, or 99% identical to a nucleic acid sequence selected         from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635,         1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480,         3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325,         5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170,         7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463,         12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046,         15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939,         16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998,         18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843,         21415-21480, 21561-21626, 21489-21552, 21635-21698 (encoding RSV         F as defined in the first aspect) and a further artificial RNA         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 9693-10052, 19853-20212, 21481, 21482,         21627, 21628, 21553, 21554, 21699, 21700 (encoding RSV M); or     -   at least one artificial RNA which is identical or at least 70%,         80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,         97%, 98%, or 99% identical to a nucleic acid sequence selected         from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635,         1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480,         3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325,         5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170,         7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463,         12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046,         15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939,         16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998,         18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843,         21415-21480, 21561-21626, 21489-21552, 21635-21698 (encoding RSV         F as defined in the first aspect) and a further artificial RNA         which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from the group         consisting of SEQ ID NOs: 10143-10502, 20222-20581, 21483,         21484, 21629, 21630, 21555, 21556, 21701, 21702 (encoding RSV         N); or     -   at least one artificial RNA which is identical or at least 70%,         80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,         97%, 98%, or 99% identical to a nucleic acid sequence selected         from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635,         1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480,         3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325,         5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170,         7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463,         12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046,         15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939,         16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998,         18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843,         21415-21480, 21561-21626, 21489-21552, 21635-21698 (encoding RSV         F as defined in the first aspect) and at least one further         artificial RNA which is identical or at least 70%, 80%, 85%,         86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,         or 99% identical to a nucleic acid sequence selected from the         group consisting of SEQ ID NOs: 10646-11005, 20591-20950, 21485,         21486, 21631, 21632, 21557, 21558, 21703, 21704 (encoding RSV         P); or     -   at least one artificial RNA which is identical or at least 70%,         80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,         97%, 98%, or 99% identical to a nucleic acid sequence selected         from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635,         1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480,         3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325,         5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170,         7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463,         12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046,         15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939,         16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998,         18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843,         21415-21480, 21561-21626, 21489-21552, 21635-21698 (encoding RSV         F as defined in the first aspect) and at least one further         artificial RNA which is identical or at least 70%, 80%, 85%,         86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,         or 99% identical to a nucleic acid sequence selected from the         group consisting of SEQ ID NOs: 11192-11551, 20960-21319, 21487,         21488, 21633, 21634, 21559, 21560, 21705, 21706 (encoding RSV         M2-1); or     -   at least one artificial RNA which is identical or at least 70%,         80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,         97%, 98%, or 99% identical to a nucleic acid sequence selected         from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635,         1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480,         3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325,         5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170,         7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463,         12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046,         15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939,         16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998,         18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843,         21415-21480, 21561-21626, 21489-21552, 21635-21698 (encoding RSV         F as defined in the first aspect) and at least one further         artificial RNA which is identical or at least 70%, 80%, 85%,         86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,         or 99% identical to a nucleic acid sequence selected from SEQ ID         NOs: 9693-10052, 19853-20212, 21481, 21482, 21627, 21628, 21553,         21554, 21699, 21700 (encoding RSV M) and a further artificial         RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from SEQ ID NOs:         10646-11005, 20591-20950, 21485, 21486, 21631, 21632, 21557,         21558, 21703, 21704 (encoding RSV P);     -   at least one artificial RNA which is identical or at least 70%,         80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,         97%, 98%, or 99% identical to a nucleic acid sequence selected         from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635,         1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480,         3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325,         5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170,         7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463,         12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046,         15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939,         16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998,         18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843,         21415-21480, 21561-21626, 21489-21552, 21635-21698 (encoding RSV         F as defined in the first aspect) and at least one further         artificial RNA which is identical or at least 70%, 80%, 85%,         86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,         or 99% identical to a nucleic acid sequence selected from SEQ ID         NOs: 9693-10052, 19853-20212, 21481, 21482, 21627, 21628, 21553,         21554, 21699, 21700 (encoding RSV M) and a further artificial         RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from SEQ ID NOs:         10646-11005, 20591-20950, 21485, 21486, 21631, 21632, 21557,         21558, 21703, 21704 (encoding RSV P) and a further artificial         RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%,         89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%         identical to a nucleic acid sequence selected from SEQ ID NOs:         10143-10502, 20222-20581, 21483, 21484, 21629, 21630, 21555,         21556, 21701, 21702 (encoding RSV N), or     -   at least one artificial RNA which is identical or at least 70%,         80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,         97%, 98%, or 99% identical to a nucleic acid sequence selected         from SEQ ID NOs: 78-86, 21415-21417, 21561-21563, 474-482,         11735-11742, 21489, 21490, 21635, 21636, 12087-12094, 493-501,         21418-21420, 21564-21566, 889-897, 12104-12111, 21491, 21492,         21637, 21638, 12456-12463, 907-914, 21421-21423, 21567-21569,         1259-1266, 12473-12480, 21493-21495, 21639-21641, 12825-12832,         1276-1283, 21424-21426, 21570-21572, 1628-1635, 12842-12849,         21496-21498, 21642-21644, 13194-13201, 1645-1652, 21433-21435,         21579-21581, 1997-2004, 13949-13956, 21505-21507, 21651-21653,         14301-14308, 2014-2021, 21436-21438, 21582-21584, 2366-2373,         14318-14325, 21508-21510, 21654-21656, 14670-14677, 2383-2390,         21439-21441, 21585-21587, 2735-2742, 14687-14694, 21511-21513,         21657-21659, 15039-15046, 2752-2759, 21442-21444, 21588-21590,         3104-3111, 15056-15063, 21514-21516, 21660-21662, 15408-15415,         3121-3128, 21445-21447, 21591-21593, 3473-3480, 15425-15432,         21517-21519, 21663-21665, 15777-15784, 3490-3497, 21448-21450,         21594-21596, 3842-3849, 15794-15801, 21520-21522, 21666-21668,         16146-16153, 3859-3866, 21427-21429, 21573-21575, 4211-4218,         13211-13218, 21499-21501, 21645-21647, 13563-13570, 4228-4235,         21430-21432, 21576-21578, 4580-4587, 13580-13587, 21502-21504,         21648-21650, 13932-13939, 4597-4604, 21451-21453, 21597-21599,         4949-4956, 16163-16170, 21523-21525, 21669-21671, 16515-16522,         4966-4973, 21454-21456, 21600-21602, 5318-5325, 16532-16539,         21526-21528, 21672-21674, 16884-16891, 5335-5342, 21457-21459,         21603-21605, 5687-5694, 16901-16908, 21529-21531, 21675-21677,         17253-17260, 5704-5711, 21460-21462, 21606-21608, 6056-6063,         17270-17277, 21532-21534, 21678-21680, 17622-17629 (encoding RSV         F as defined in the first aspect) and at least one further         artificial RNA which is identical or at least 70%, 80%, 85%,         86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,         or 99% identical to a nucleic acid sequence selected from the         group consisting of SEQ ID NOs: 11192-11551, 20960-21319, 21487,         21488, 21633, 21634, 21559, 21560, 21705, 21706 (encoding RSV         M2-1),

wherein, optionally, at least one or more than one, or wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.

In various embodiments, different combinations of RSV F (F0, F-del, F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0, F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1_mut3, F_DSCav1_mut4, F-del_DSCav1_mut4, F_DSCav1_mut5, F-del_DSCav1_mut5, F_DSCav1_mut6, F-del_DSCav1_mut6, F_DSCav1_mut7, F-del_DSCav1_mut7, F_DSCav1_mut8, F-del_DSCav1_mut8) RNA constructs and RSV T-cell antigen RNA constructs (RSV M, N, M2-1, or P) are suitably comprised in the composition (as disclosed in Table 7B; combinations 1-64). The combinations 49-64 are preferred, combination 64 is particularly preferred.

TABLE 7B Suitable combinations of RNA constructs encoding RSV F and RNA constructs encoding T-cell antigens SEQ ID NO: T-cell antigen SEQ ID NO: Combination RSV F construct Protein construct Protein 1 F0 68 M 9684 2 F-del 483 M 9684 3 F_DSCav1 898 M 9684 4 F-del_DSCav1 1267 M 9684 5 F_DSCav1_mut1 1636 M 9684 6 F-del_DSCav1_mut1 2005 M 9684 7 F_DSCav1_mut2 2374 M 9684 8 F-del_DSCav1_mut2 2743 M 9684 9 F_DSCav1_mut3 3112 M 9684 10 F-del_DSCav1_mut3 3481 M 9684 11 F_DSCav1_mut0 3850 M 9684 12 F-del_DSCav1_mut0 4219 M 9684 13 F_DSCav1_mut4 4588 M 9684 14 F-del_DSCav1_mut4 4957 M 9684 15 F_DSCav1_mut5 5326 M 9684 16 F-del_DSCav1_mut5 5695 M 9684 17 F0 68 N 10134 18 F-del 483 N 10134 19 F_DSCav1 898 N 10134 20 F-del_DSCav1 1267 N 10134 21 F_DSCav1_mut1 1636 N 10134 22 F-del_DSCav1_mut1 2005 N 10134 23 F_DSCav1_mut2 2374 N 10134 24 F-del_DSCav1_mut2 2743 N 10134 25 F_DSCav1_mut3 3112 N 10134 26 F-del_DSCav1_mut3 3481 N 10134 27 F_DSCav1_mut0 3850 N 10134 28 F-del_DSCav1_mut0 4219 N 10134 29 F_DSCav1_mut4 4588 N 10134 30 F-del_DSCav1_mut4 4957 N 10134 31 F_DSCav1_mut5 5326 N 10134 32 F-del_DSCav1_mut5 5695 N 10134 33 F0 68 P 10637 34 F-del 483 P 10637 35 F_DSCav1 898 P 10637 36 F-del_DSCav1 1267 P 10637 37 F_DSCav1_mut1 1636 P 10637 38 F-del_DSCav1_mut1 2005 P 10637 39 F_DSCav1_mut2 2374 P 10637 40 F-del_DSCav1_mut2 2743 P 10637 41 F_DSCav1_mut3 3112 P 10637 42 F-del_DSCav1_mut3 3481 P 10637 43 F_DSCav1_mut0 3850 P 10637 44 F-del_DSCav1_mut0 4219 P 10637 45 F_DSCav1_mut4 4588 P 10637 46 F-del_DSCav1_mut4 4957 P 10637 47 F_DSCav1_mut5 5326 P 10637 48 F-del_DSCav1_mut5 5695 P 10637 49 F0 68 M2-1 11183 50 F-del 483 M2-1 11183 51 F_DSCav1 898 M2-1 11183 52 F-del_DSCav1 1267 M2-1 11183 53 F_DSCav1_mut1 1636 M2-1 11183 54 F-del_DSCav1_mut1 2005 M2-1 11183 55 F_DSCav1_mut2 2374 M2-1 11183 56 F-del_DSCav1_mut2 2743 M2-1 11183 57 F_DSCav1_mut3 3112 M2-1 11183 58 F-del_DSCav1_mut3 3481 M2-1 11183 59 F_DSCav1_mut0 3850 M2-1 11183 60 F-del_DSCav1_mut0 4219 M2-1 11183 61 F_DSCav1_mut4 4588 M2-1 11183 62 F-del_DSCav1_mut4 4957 M2-1 11183 63 F_DSCav1_mut5 5326 M2-1 11183 64 F-del_DSCav1_mut5 5695 M2-1 11183

Table 7B discloses compositions of the second aspect comprising at least one RNA encoding RSV F from the strain A2 as defined in the first aspect and at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV from the strain A2 selected from matrix protein M, nucleoprotein N, M2-1 protein, phosphoprotein P. In further embodiments of the invention all disclosed compositions in Table 7B are also applicable for antigenic peptides or proteins derived from a RSV isolate Memphis-37 (strain Memphis-37).

In a particularly preferred embodiment, the composition comprises one RSV F RNA construct (F0, F-del, F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0, F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1_mut3, F_DSCav1_mut4, F-del_DSCav1_mut4, F_DSCav1_mut5, F-del_DSCav1_mut5, F_DSCav1_mut6, F-del_DSCav1_mut6, F_DSCav1_mut7, F-del_DSCav1_mut7, F_DSCav1_mut8, F-del_DSCav1_mut8) and, in addition, one RSV M2-1 RNA construct, preferably F-del_DSCav1_mut5 and M2-1.

In particularly preferred embodiments, one RNA construct encoding RSV F, selected from SEQ ID NOs: 5704-5711, 21460-21462, 21606-21608, 17270-17277, 21532-21534, 21678-21680 and one RNA construct encoding M2-1, selected from SEQ ID NOs: 11192-11199, 21487, 21488, 21633, 21634, 20960-20967, 21559, 21560, 21705, 21706 are comprised in the composition of the invention.

In various embodiments, the at least one artificial RNA of the first aspect and the at least one further artificial RNA as specified herein are derived from the same RSV virus (e.g. any virus selected from List 1).

In preferred embodiments, the at least one artificial RNA of the first aspect is derived from HRSV(A2) and the at least one further artificial RNA as specified herein is derived from HRSV(A2).

In preferred embodiments, the at least one artificial RNA of the first aspect is derived from HRSV(Memphis-37) and the at least one further artificial RNA as specified herein is derived from HRSV(Memphis-37).

It has to be understood that in the context of the invention, certain combinations of coding sequences may be generated by any combination of monocistronic, bicistronic and multicistronic artificial nucleic acids and/or multi-antigen-constructs/nucleic acid to obtain a nucleic acid composition encoding multiple antigenic peptides or proteins as defined herein.

Furthermore, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be used as well, which are suitable for administration to a person. The term “compatible” as used herein means that the constituents of the composition are capable of being mixed with the at least one RNA and, optionally, the further artificial RNA of the composition, in such a manner that no interaction occurs, which would substantially reduce the biological activity or the pharmaceutical effectiveness of the composition under typical use conditions. Pharmaceutically acceptable carriers, fillers and diluents must have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated. Compounds which may be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as, for example, lactose, glucose, trehalose and sucrose; starches, such as, for example, corn starch or potato starch; dextrose; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

Further additives, which may be included in the composition are emulsifiers, such as, for example, Tween; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; preservatives.

Complexation:

In a preferred embodiment of the second aspect, the at least one artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed or associated with or at least partially complexed or partially associated with one or more cationic or polycationic compound, preferably cationic or polycationic polymer, cationic or polycationic polysaccharide, cationic or polycationic lipid, cationic or polycationic protein, cationic or polycationic peptide, or any combinations thereof.

The term “cationic or polycationic compound” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a charged molecule, which is positively charged at a pH value ranging from about 1 to 9, at a pH value ranging from about 3 to 8, at a pH value ranging from about 4 to 8, at a pH value ranging from about 5 to 8, more preferably at a pH value ranging from about 6 to 8, even more preferably at a pH value ranging from about 7 to 8, most preferably at a physiological pH, e.g. ranging from about 7.2 to about 7.5. Accordingly, a cationic component, e.g. a cationic peptide, cationic protein, cationic polymer, cationic polysaccharide, cationic lipid may be any positively charged compound or polymer which is positively charged under physiological conditions. A “cationic or polycationic peptide or protein” may contain at least one positively charged amino acid, or more than one positively charged amino acid, e.g. selected from Arg, His, Lys or Orn. Accordingly, “polycationic” components are also within the scope exhibiting more than one positive charge under the given conditions.

Cationic or polycationic compounds, being particularly preferred in this context may be selected from the following list of cationic or polycationic peptides or proteins of fragments thereof: protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides, pAntp, ps, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, or histones. More preferably, the nucleic acid as defined herein, preferably the mRNA as defined herein, is complexed with one or more polycations, preferably with protamine or oligofectamine, most preferably with protamine.

In a preferred embodiment of the second aspect, the at least one artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with protamine

Further preferred cationic or polycationic compounds, which can be used as transfection or complexation agent may include cationic polysaccharides, for example chitosan, polybrene etc.; cationic lipids, e.g. DOTMA, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS, DIMRI, DOTAP, DC-6-14, CLIP1, CLIP6, CLIP9, oligofectamine; or cationic or polycationic polymers, e.g. modified polyaminoacids, such as beta-aminoacid-polymers or reversed polyamides, etc., modified polyethylenes, such as PVP etc., modified acrylates, such as pDMAEMA etc., modified amidoamines such as pAMAM etc., modified polybetaaminoester (PBAE), such as diamine end modified 1,4 butanediol diacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such as polypropylamine dendrimers or pAMAM based dendrimers, etc., polyimine(s), such as PEI, poly(propyleneimine), etc., polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based polymers, etc., silan backbone based polymers, such as PMOXA-PDMS copolymers, etc., blockpolymers consisting of a combination of one or more cationic blocks (e.g. selected from a cationic polymer as mentioned above) and of one or more hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.

In this context it is particularly preferred that the at least one artificial RNA as defined herein and, optionally, the further artificial RNA of the second aspect, is complexed or at least partially complexed with a cationic or polycationic compound and/or a polymeric carrier, preferably cationic proteins or peptides. In this context, the disclosure of WO2010/037539 and WO2012/113513 is incorporated herewith by reference. Partially means that only a part of the artificial nucleic acid is complexed with a cationic compound and that the rest of the artificial nucleic acid is (comprised in the inventive (pharmaceutical) composition) in uncomplexed form (“free”).

In a preferred embodiment of the second aspect, the at least one artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with one or more cationic or polycationic compounds, preferably protamine, and at least one free artificial RNA of the first aspect and, optionally the further RNA of the second aspect.

In this context it is particularly preferred that the at least one artificial RNA as defined herein, and, optionally, the further artificial RNA of the second aspect, is complexed, or at least partially complexed with protamine.

Preferably, the molar ratio of the nucleic acid, particularly the RNA of the protamine-complexed RNA to the free RNA may be selected from a molar ratio of about 0.001:1 to about 1:0.001, including a ratio of about 1:1. Suitably, the complexed RNA is complexed with protamine by addition of protamine-trehalose solution to the RNA sample at a RNA:protamine weight to weight ratio (w/w) of 2:1.

Further preferred cationic or polycationic proteins or peptides that may be used for complexation can be derived from formula (Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x of the patent application WO2009/030481 or WO2011/026641, the disclosure of WO2009/030481 or WO2011/026641 relating thereto incorporated herewith by reference.

In a preferred embodiment of the second aspect, the at least one artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed, or at least partially complexed with at least one cationic or polycationic proteins or peptides preferably selected from SEQ ID NOs: 64-67, 21320 or any combinations thereof.

According to embodiments, the composition of the present invention comprises the RNA as defined herein, and a polymeric carrier.

The term “polymeric carrier” as used herein will be recognized and understood by the person of ordinary skill in the art, and are for example intended to refer to a compound that facilitates transport and/or complexation of another compound (cargo). A polymeric carrier is typically a carrier that is formed of a polymer. A polymeric carrier may be associated to its cargo (nucleic acid, RNA) by covalent or non-covalent interaction.

A suitable polymeric carrier may be a polymeric carrier formed by disulfide-crosslinked cationic compounds. The disulfide-crosslinked cationic compounds may be the same or different from each other. The polymeric carrier can also contain further components. The polymeric carrier used according to the present invention may comprise mixtures of cationic peptides, proteins or polymers and optionally further components as defined herein, which are crosslinked by disulfide bonds (via —SH groups).

In this context, polymeric carriers according to formula {(Arg)I;(Lys)m;(His)n;(Orn)o;(Xaa′)x(Cys)y} and formula Cys,{(Arg);(Lys)m;(His)n;(Orn)o;(Xaa)x}Cys2 of the patent application WO2012/013326 are preferred, the disclosure of WO2012/013326 relating thereto incorporated herewith by reference.

In embodiments, the polymeric carrier used to complex the RNA as defined herein may be derived from a polymeric carrier molecule according formula (L-P¹-S-[S-P²-S]_(n)-S-P¹-L) of the patent application WO2011/026641, the disclosure of WO2011/026641 relating thereto incorporated herewith by reference.

In embodiments, the polymeric carrier compound is formed by, or comprises or consists of the peptide elements CysArg12Cys (SEQ ID NO: 64) or CysArg12 (SEQ ID NO: 65) or TrpArg12Cys (SEQ ID NO: 66). In particularly preferred embodiments, the polymeric carrier compound consists of a (R₁₂C)-(R₁₂C) dimer, a (WR₁₂C)-(WR₁₂C) dimer, or a (CR₁₂)-(CR₁₂C)-(CR₁₂) trimer, wherein the individual peptide elements in the dimer (e.g. (WR₁₂C)), or the trimer (e.g. (CR₁₂)), are connected via —SH groups.

In a preferred embodiment of the second aspect, the at least one artificial RNA of the first aspect and optionally the further artificial RNA of the second aspect, is complexed or associated with a polyethylene glycol/peptide polymer comprising HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)7-S-PEG5000-OH (SEQ ID NO: 67 as peptide monomer).

In a further preferred embodiment of the second aspect, the at least one artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed or associated with a polyethylene glycol/peptide polymer comprising HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S-)4-S-PEG5000-OH (SEQ ID NO: 67 as peptide monomer).

In a further preferred embodiment of the second aspect, the at least one artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed or associated with a polyethylene glycol/peptide polymer comprising HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)7-S-PEG5000-OH (SEQ ID NO: 21320 as peptide monomer).

In a further preferred embodiment of the second aspect, the at least one artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed or associated with a polyethylene glycol/peptide polymer comprising HO-PEG5000-S-(S-CGHHHHHRRRRHHHHHGC-S-)4-S-PEG5000-OH (SEQ ID NO: 21320 as peptide monomer).

In other embodiments, the composition comprises at least one artificial RNA as described herein, and, optionally, the further artificial RNA of the second aspect, wherein the at least one artificial RNA and, optionally, the further artificial RNA of the second aspect, is complexed or associated with polymeric carriers and, optionally, with at least one lipid component as described in the published PCT applications WO2017/212008A1, WO2017/212006A1, WO2017/212007A1, and WO2017/212009A1. In this context, the disclosures of WO2017/212008A1, WO2017/212006A1, WO2017/212007A1, and WO2017/212009A1 are herewith incorporated by reference.

In a particularly preferred embodiment, the polymeric carrier is a peptide polymer, preferably a polyethylene glycol/peptide polymer as defined above, and a lipid component, preferably a lipidoid component, more preferably lipidoid component.

In preferred embodiment of the second aspect, the at least one artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed or associated with a polymeric carrier, preferably with a polyethylene glycol/peptide polymer as defined above, and a lipidoid component, wherein the lipidoid component is a compound according to formula A

wherein

-   -   R_(A) is independently selected for each occurrence an         unsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀         aliphatic group; a substituted or unsubstituted, cyclic or         acyclic, branched or unbranched C₁₋₂₀ heteroaliphatic group; a         substituted or unsubstituted aryl; a substituted or         unsubstituted heteroaryl;

wherein at least one R_(A) is

-   -   R₅ is independently selected for each occurrence of from an         unsubstituted, cyclic or acyclic, branched or unbranched C₈₋₁₆         aliphatic; a substituted or unsubstituted aryl; or a substituted         or unsubstituted heteroaryl;     -   each occurrence of x is an integer from 1 to 10;     -   each occurrence of y is an integer from 1 to 10;

or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the lipidoid component is 3-C12-OH according to formula B

In preferred embodiments, the peptide polymer comprising lipidoid 3-C12-OH as specified above is used to complex the RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, to form complexes having an N/P ratio from about 0.1 to about 20, or from about 0.2 to about 15, or from about 2 to about 15, or from about 2 to about 12, wherein the N/P ratio is defined as the mole ratio of the nitrogen atoms of the basic groups of the cationic peptide or polymer to the phosphate groups of the artificial nucleic acid. In that context, the disclosure of WO2017/212009A1, in particular Claims 1 to 10 of WO2017/212009A1, and the specific disclosure relating thereto is herewith incorporated by reference.

Encapsulation/Complexation in LNPs:

In preferred embodiments of the second aspect, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed or associated with one or more lipids (e.g. cationic lipids and/or neutral lipids), thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and/or nanoliposomes.

For compositions comprising more than one artificial RNA construct as defined herein (e,g, F-del and M2-1), said constructs may be co-formulated in e.g. LNPs to form the respective composition.

Alternatively, said more than one artificial RNA constructs may be formulated separately, and may subsequently be combined, to form the respective composition.

In this context, the terms “complexed” or “associated” refer to the essentially stable combination of artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, with one or more lipids into larger complexes or assemblies without covalent binding.

The term “lipid nanoparticle”, also referred to as “LNP”, is not restricted to any particular morphology, and include any morphology generated when a cationic lipid and optionally one or more further lipids are combined, e.g. in an aqueous environment and/or in the presence of RNA. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle (LNP).

Accordingly, in preferred embodiments of the second aspect, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with one or more lipids thereby forming lipid nanoparticles (LNP).

LNPs typically comprise a cationic lipid and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g. PEGylated lipid). The RNA may be encapsulated in the lipid portion of the LNP or an aqueous space enveloped by some or the entire lipid portion of the LNP. The RNA or a portion thereof may also be associated and complexed with the LNP. An LNP may comprise any lipid capable of forming a particle to which the nucleic acids are attached, or in which the one or more nucleic acids are encapsulated.

Preferably, the LNP comprising nucleic acids comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and PEGylated lipids.

The cationic lipid of an LNP may be cationisable, i.e. it becomes protonated as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

The LNP may comprise any further cationic or cationisable lipid, i.e. any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH.

Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethyl ammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DM A), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 Ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 Ether), LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the foregoing.

In some embodiments, the lipid is selected from the group consisting of 98N12-5, C12-200, and ckk-E12.

In one embodiment, the further cationic lipid is an amino lipid.

Representative amino lipids include, but are not limited to, 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US20100324120).

In one embodiment, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, may be formulated in an aminoalcohol lipidoid. Aminoalcohol lipidoids which may be used in the present invention may be prepared by the methods described in U.S. Pat. No. 8,450,298, herein incorporated by reference in its entirety. Suitable (ionizable) lipids can also be the compounds as disclosed in Tables 1, 2 and 3 and as defined in claims 1-24 of WO2017/075531A1, hereby incorporated by reference.

In another embodiment, ionizable lipids can also be the compounds as disclosed in WO2015/074085A1 (i.e. ATX-001 to ATX-032 or the compounds as specified in claims 1-26), U.S. Appl. Nos. 61/905,724 and 15/614,499 or U.S. Pat. Nos. 9,593,077 and 9,567,296 hereby incorporated by reference in their entirety.

In that context, any lipid derived from generic formula (X1)

wherein, Ri and R2 are the same or different, each a linear or branched alkyl consisting of 1 to 9 carbons, an alkenyl or alkynyl consisting of 2 to 11 carbons, Li and L2 are the same or different, each a linear alkylene or alkenylene consisting of 5 to 18 carbons, or forming a heterocycle with N, Xi is a bond, or is —CO—O— whereby -L2-CO—O—R2 is formed, X2 is S or O, L3 is a bond or a linear or branched alkylene consisting of 1 to 6 carbons, or forming a heterocycle with N, R3 is a linear or branched alkylene consisting of 1 to 6 carbons, and R4 and R 5 are the same or different, each hydrogen or a linear or branched alkyl consisting of 1 to 6 carbons; or a pharmaceutically acceptable salt thereof may be suitably used.

In other embodiments, suitable cationic lipids can also be the compounds as disclosed in WO2017/117530A1 (i.e. lipids 13, 14, 15, 16, 17, 18, 19, 20, or the compounds as specified in the claims), hereby incorporated by reference in its entirety.

In that context, any lipid derived from generic formula (X2)

wherein

X is a linear or branched alkylene or alkenylene, monocyclic, bicyclic, or tricyclic arene or heteroarene;

Y is a bond, an ethene, or an unsubstituted or substituted aromatic or heteroaromatic ring; Z is S or O;

L is a linear or branched alkylene of 1 to 6 carbons;

R-3 and R4 are independently a linear or branched alkyl of 1 to 6 carbons;

Ri and R2 are independently a linear or branched alkyl or alkenyl of 1 to 20 carbons; r is 0 to 6; and

m, n, p, and q are independently 1 to 18;

wherein when n=q, m=p, and Ri=R2, then X and Y differ;

wherein when X=Y, n=q, m=p, then Ri and R2 differ;

wherein when X=Y, n=q, and Ri=R2, then m and p differ; and

wherein when X=Y, m=p, and Ri=R2, then n and q differ;

or a pharmaceutically acceptable salt thereof.

In preferred embodiments, a lipid may be used derived from formula (X2), wherein, X is a bond, linear or branched alkylene, alkenylene, or monocyclic, bicyclic, or tricyclic arene or heteroarene; Y is a monocyclic, bicyclic, or tricyclic arene or heteroarene; Z is S or O; L is a linear or branched alkylene of 1 to 6 carbons; R3 and R4 are independently a linear or branched alkyl of 1 to 6 carbons; Ri and R2 are independently a linear or branched alkyl or alkenyl of 1 to 20 carbons; r is 0 to 6; and m, n, p, and q are independently 1 to 18; or a pharmaceutically acceptable salt thereof may be suitably used.

In preferred embodiments, ionizable lipids may also be selected from the lipid compounds disclosed in PCT application PCT/EP2017/077517 (i.e. lipid compounds derived form formula I, II, and III of PCT/EP2017/077517, or lipid compounds as specified in Claims 1 to 12 of PCT/EP2017/077517), the disclosure of PCT/EP2017/077517 hereby incorporated by reference in its entirety. In that context, lipid compounds disclosed in Table 7 of PCT/EP2017/077517 (e.g. lipid compounds derived from formula I-1 to I-41) and lipid compounds disclosed in Table 8 of PCT/EP2017/077517 (e.g. lipid compounds derived from formula I-1 to II-36) may be suitably used in the context of the invention. Accordingly, formula I-1 to formula I-41 and formula II-1 to formula I-36 of PCT/EP2017/077517, and the specific disclosure relating thereto, are herewith incorporated by reference.

In particularly preferred embodiments of the second aspect, a suitable lipid may be a cationic lipid according to formula (III)

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein, R1, R2, R3, L1, L2, G1, G2, and G3 are as below.

Formula (III) is further defined in that:

one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other of L1 or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O— or a direct bond;

G¹ and G² are each independently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene;

G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈ cycloalkenylene;

R^(a) is H or C₁-C₁₂ alkyl;

R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;

R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴;

R⁴ is C₁-C₁₂ alkyl;

R⁵ is H or C₁-C₁₂ alkyl; and

x is 0, 1 or 2.

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

wherein:

A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; R⁶ is, at each occurrence, independently H, OH or C₁-C₂₄ alkyl; n is an integer ranging from 1 to 15.

In some of the foregoing embodiments of formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

In other embodiments of formula (III), the lipid has one of the following structures (IIC) or (IIID):

wherein y and z are each independently integers ranging from 1 to 12.

In any of the foregoing embodiments of formula (III), one of L¹ or L² is —O(C═O)—. For example, in some embodiments each of L¹ and L² are —O(C═O)—. In some different embodiments of any of the foregoing, L¹ and L² are each independently —(C═O)O— or —O(C═O)—. For example, in some embodiments each of L¹ and L² is —(C═O)O—.

In preferred embodiments of the second aspect, the cationic lipid of the LNP is a compound of formula I, wherein:

L¹ and L² are each independently —O(C═O)— or (C═O)—O—;

G³ is C₁-C₂₄ alkylene or C₁-C₂₄ alkenylene; and

R³ is H or OR⁵.

In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIG), (IIIH),

In some of the foregoing embodiments of formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. In some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some other of the foregoing embodiments of formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6. In some of the foregoing embodiments of formula (II), R⁶ is H. In other of the foregoing embodiments, R⁶ is C₁-C₂₄ alkyl. In other embodiments, R⁶ is OH. In some embodiments of formula (III), G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G³ is linear C₁-C₂₄ alkylene or linear C₁-C₂₄ alkenylene. In some other foregoing embodiments of formula (III), R¹ or R², or both, is C₆-C₂₄ alkenyl. For example, in some embodiments, R¹ and R² each, independently have the following structure:

wherein:

R^(7a) and R^(7b) are, at each occurrence, independently H or C₁-C₁₂ alkyl; and a is an integer from 2 to 12, wherein

R^(7a), R^(7b) and a are each selected such that R¹ and R² each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12. In some of the foregoing embodiments of formula (III), at least one occurrence of R^(7a) is H. For example, in some embodiments, R^(7a) is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R^(7b) is C₁-C₈ alkyl. For example, in some embodiments, C₁-C₈ alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In different embodiments of formula (III), R¹ or R², or both, has one of the following structures:

In preferred embodiments of the second aspect, the cationic lipid of the LNP is a compound of formula III, wherein:

L¹ and L² are each independently —O(C═O)— or (C═O)—O—; and

R¹ and R² each independently have one of the following structures:

In some of the foregoing embodiments of formula (III), R³ is OH, CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NHC(═O)R⁴. In some embodiments, R⁴ is methyl or ethyl.

In preferred embodiments of the second aspect, the cationic lipid of the LNP is a compound of formula II, wherein R³ is OH.

In particularly preferred embodiment of the second aspect, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein the LNP is selected from structures III-1 to III-36 (see Table 8).

TABLE 8 Representative lipid compounds derived from formula (III) No. Structure III-1

III-2

III-3

III-4

III-5

III-6

III-7

III-8

III-9

III-10

III-11

III-12

III-13

III-14

III-15

III-16

III-17

III-18

III-19

III-20

III-21

III-22

III-23

III-24

III-25

III-26

III-27

III-28

III-29

III-30

III-31

III-32

III-33

III-34

III-35

III-36

In some embodiments, the LNPs comprise a lipid of formula (III), an artificial RNA of the first aspect, and, optionally, the further artificial RNA of the second aspect, and one or more excipient selected from neutral lipids, steroids and PEGylated lipids. In some embodiments the lipid of formula (III) is compound III-3. In some embodiments the lipid of formula (III) is compound III-7.

In preferred embodiments, the LNP comprises a cationic lipid selected from:

In particularly preferred embodiment of the second aspect, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein the LNP comprises the following cationic lipid (lipid according to formula III-3 of Table 8):

In certain embodiments, the cationic lipid as defined herein, preferably as disclosed in Table 8, more preferably cationic lipid compound III-3, is present in the LNP in an amount from about 30 to about 95 mole percent, relative to the total lipid content of the LNP. If more than one cationic lipid is incorporated within the LNP, such percentages apply to the combined cationic lipids.

In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent, such as about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mole percent, respectively. In embodiments, the cationic lipid is present in the LNP in an amount from about 47 to about 48 mole percent, such as about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 50.0 mole percent, respectively, wherein 47.7 mole percent are particularly preferred.

In some embodiments, the cationic lipid is present in a ratio of from about 20 mol % to about 70 or 75 mol % or from about 45 to about 65 mol % or about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or about 70 mol % of the total lipid present in the LNP. In further embodiments, the LNPs comprise from about 25% to about 75% on a molar basis of cationic lipid, e.g., from about 20 to about 70%, from about 35 to about 65%, from about 45 to about 65%, about 60%, about 57.5%, about 57.1%, about 50% or about 40% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle). In some embodiments, the ratio of cationic lipid to nucleic acid, preferably to the artificial RNA of the first aspect, and, optionally, the further artificial RNA of the second aspect, is from about 3 to about 15, such as from about 5 to about 13 or from about 7 to about 11.

In some embodiments of the invention the LNP comprises a combination or mixture of any the lipids described above.

Other suitable (cationic) lipids are disclosed in WO2009/086558, WO2009/127060, WO2010/048536, WO2010/054406, WO2010/088537, WO2010/129709, WO2011/153493, US2011/0256175, US2012/0128760, US2012/0027803, U.S. Pat. No. 8,158,601, WO2016/118724, WO2016/118725, WO2017/070613, WO2017/070620, WO2017/099823, and WO2017/112865. In that context, the disclosures of WO2009/086558, WO2009/127060, WO2010/048536, WO2010/054406, WO2010/088537, WO2010/129709, WO2011/153493, US2011/0256175, US2012/0128760, US2012/0027803, U.S. Pat. No. 8,158,601, WO2016/118724, WO2016/118725, WO2017/070613, WO2017/070620, WO2017/099823, and WO2017/112865 specifically relating to (cationic) lipids suitable for LNPs are incorporated herewith by reference.

In some embodiments, the lipid is selected from the group consisting of 98N12-5, C12-200, and ckk-E12.

In some embodiments, amino or cationic lipids as defined herein have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g. pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise suitable in the context of the present invention.

In some embodiments, the protonatable lipids have a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.

LNPs can comprise two or more (different) cationic lipids. The cationic lipids may be selected to contribute different advantageous properties. For example, cationic lipids that differ in properties such as amine pKa, chemical stability, half-life in circulation, half-life in tissue, net accumulation in tissue, or toxicity can be used in the LNP. In particular, the cationic lipids can be chosen so that the properties of the mixed-LNP are more desirable than the properties of a single-LNP of individual lipids.

The amount of the permanently cationic lipid or lipidoid may be selected taking the amount of the nucleic acid cargo into account. In one embodiment, these amounts are selected such as to result in an N/P ratio of the nanoparticle(s) or of the composition in the range from about 0.1 to about 20. In this context, the N/P ratio is defined as the mole ratio of the nitrogen atoms (“N”) of the basic nitrogen-containing groups of the lipid or lipidoid to the phosphate groups (“P”) of the RNA which is used as cargo. The N/P ratio may be calculated on the basis that, for example, 1 μg RNA typically contains about 3 nmol phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The “N”-value of the lipid or lipidoid may be calculated on the basis of its molecular weight and the relative content of permanently cationic and—if present—cationisable groups.

LNP in vivo characteristics and behavior can be modified by addition of a hydrophilic polymer coating, e.g. polyethylene glycol (PEG), to the LNP surface to confer steric stabilization. Furthermore, LNPs can be used for specific targeting by attaching ligands (e.g. antibodies, peptides, and carbohydrates) to its surface or to the terminal end of the attached PEG chains (e.g. via PEGylated lipids).

In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a PEGylated lipid. The term “PEGylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG) and the like.

In certain embodiments, the LNP comprises an additional, stabilizing-lipid which is a polyethylene glycol-lipid (PEGylated lipid). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g. PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In a preferred embodiment, the polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a PEGylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as w-methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(w-methoxy(polyethoxy)ethyl)carbamate.

In preferred embodiments of the second aspect, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein the LNP additionally comprises a PEGylated lipid with the formula (IV):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has mean value ranging from 30 to 60.

In some of the foregoing embodiments of the PEGylated lipid according to formula (IV), R⁸ and R⁹ are not both n-octadecyl when w is 42. In some other embodiments, R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R⁸ is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R⁹ is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

In various embodiments, w spans a range that is selected such that the PEG portion of the PEGylated lipid according to formula (IV) has an average molecular weight of about 400 to about 6000 g/mol. In some embodiments, the average w is about 50.

In preferred embodiments of the second aspect, R⁸ and R⁹ of the PEGylated lipid according to formula (IV) are saturated alkyl chains.

In a particularly preferred embodiment of the second aspect, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein the LNP additionally comprises a PEGylated lipid, wherein the PEG lipid is of formula (IVa)

wherein n has a mean value ranging from 30 to 60, such as about 28 to about 32, about 30 to about 34, 32 to about 36, about 34 to about 38, 36 to about 40, about 38 to about 42, 40 to about 44, about 42 to about 46, 44 to about 48, about 46 to about 50, 48 to about 52, about 50 to about 54, 52 to about 56, about 54 to about 58, 56 to about 60, about 58 to about 62. In preferred embodiments, n is about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54. In a most preferred embodiment n has a mean value of 49.

In other embodiments, the PEGylated lipid has one of the following structures:

wherein n is an integer selected such that the average molecular weight of the PEGylated lipid is about 2500 g/mol, most preferably n is about 49.

Further examples of PEG-lipids suitable in that context are provided in US2015/0376115A1 and WO2015/199952, each of which is incorporated by reference in its entirety.

In some embodiments, LNPs include less than about 3, 2, or 1 mole percent of PEG or PEG-modified lipid, based on the total moles of lipid in the LNP. In further embodiments, LNPs comprise from about 0.1% to about 20% of the PEG-modified lipid on a molar basis, e.g., about 0.5 to about 10%, about 0.5 to about 5%. about 10%. about 5%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.5%, or about 0.3% on a molar basis (based on 100% total moles of lipids in the LNP). In preferred embodiments, LNPs comprise from about 1.0% to about 2.0% of the PEG-modified lipid on a molar basis, e.g., about 1.2 to about 1.9%, about 1.2 to about 1.8%, about 1.3 to about 1.8%, about 1.4 to about 1.8%, about 1.5 to about 1.8%, about 1.6 to about 1.8%, in particular about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, most preferably 1.7% (based on 100% total moles of lipids in the LNP). In various embodiments, the molar ratio of the cationic lipid to the PEGylated lipid ranges from about 100:1 to about 25:1.

In preferred embodiments, the LNP additionally comprises one or more additional lipids which stabilize the formation of particles during their formation (e.g. neutral lipid and/or one or more steroid or steroid analogue).

In preferred embodiments of the second aspect, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein the LNP additionally comprises one or more neutral lipid and/or one or more steroid or steroid analogue.

Suitable stabilizing lipids include neutral lipids and anionic lipids. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

In embodiments of the second aspect, the LNP additionally comprises one or more neutral lipids, wherein the neutral lipid is selected from the group comprising distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).

In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2:1 to about 8:1.

In preferred embodiments of the second aspect, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). The molar ratio of the cationic lipid to DSPC may be in the range from about 2:1 to 8:1.

In preferred embodiments of the second aspect, the steroid is cholesterol. The molar ratio of the cationic lipid to cholesterol may be in the range from about 2:1 to 1:1.

The sterol can be about 10 mol % to about 60 mol % or about 25 mol % to about 40 mol % of the lipid particle. In one embodiment, the sterol is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 mol % of the total lipid present in the lipid particle. In another embodiment, the LNPs include from about 5% to about 50% on a molar basis of the sterol, e.g., about 15% to about 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%, about 35%, about 34.4%, about 31.5% or about 31% on a molar basis (based upon 100% total moles of lipid in the lipid nanoparticle).

Preferably, lipid nanoparticles (LNPs) comprise: (a) at least one artificial RNA of the first aspect, and, optionally, the further artificial RNA of the second aspect, (b) a cationic lipid, (c) an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG-modified lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and (e) optionally, a sterol.

In other preferred embodiments, lipid nanoparticles (LNPs) comprise: (a) at least one artificial RNA encoding F of the first aspect or a derivative or fragment thereof and at least one artificial RNA encoding N, M, P, or M2-1 of the second aspect or a derivative or fragment thereof, (b) a cationic lipid, (c) an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG-modified lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and (e) optionally, a sterol.

In some embodiments, the LNPs comprise a lipid of formula (III), an artificial RNA as defined herein, a neutral lipid, a steroid and a PEGylated lipid. In preferred embodiments, the lipid of formula (III) is lipid compound III-3, the neutral lipid is DSPC, the steroid is cholesterol, and the PEGylated lipid is the compound of formula (IVa).

In a preferred embodiment of the second aspect, the LNP consists essentially of (i) at least one cationic lipid; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g. PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.

In particularly preferred embodiments of the second aspect, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein the LNP essentially consists of

(i) at least one cationic lipid as defined herein, preferably a lipid of formula (III), more preferably lipid III-3; (ii) a neutral lipid as defined herein, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (iii) a steroid or steroid analogue as defined herein, preferably cholesterol; and (iv) a PEG-lipid as defined herein, e.g. PEG-DMG or PEG-cDMA, preferably a PEGylated lipid of formula (IVa), wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.

In one preferred embodiment, the lipid nanoparticle comprises: a cationic lipid with formula (III) and/or PEG lipid with formula (IV), optionally a neutral lipid, preferably 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and optionally a steroid, preferably cholesterol, wherein the molar ratio of the cationic lipid to DSPC is optionally in the range from about 2:1 to 8:1, wherein the molar ratio of the cationic lipid to cholesterol is optionally in the range from about 2:1 to 1:1.

In a particular preferred embodiment, the composition of the second aspect comprising the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, comprises lipid nanoparticles (LNPs), which have a molar ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e. proportion (mol %) of cationic lipid (preferably lipid III-3), DSPC, cholesterol and PEG-lipid ((preferably PEG-lipid of formula (IVa) with n=49); solubilized in ethanol).

In a particular preferred embodiment, the composition of the second aspect comprises lipid nanoparticles (LNPs), which have a molar ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e. proportion (mol %) of cationic lipid (preferably lipid III-3), DSPC, cholesterol and PEG-lipid ((preferably PEG-lipid of Formula (IVa) with n=49); solubilized in ethanol), wherein the lipid nanoparticles comprise at least one RNA encoding F (F0, F-del, F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0, F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1_mut3, F_DSCav1_mut4, F-del_DSCav1_mut4, F_DSCav1_mut5, F-del_DSCav1_mut5, F_DSCav1_mut6, F-del_DSCav1_mut6, F_DSCav1_mut7, F-del_DSCav1_mut7, F_DSCav1_mut8, or F-del_DSCav1_mut8), and, additionally lipid nanoparticles (LNPs), which have a molar ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e. proportion (mol %) of cationic lipid (preferably lipid III-3), DSPC, cholesterol and PEG-lipid ((preferably PEG-lipid of Formula (IVa) with n=49); solubilized in ethanol), wherein the lipid nanoparticles comprise at least one RNA encoding M, N, M2-1, or P.

In a particular preferred embodiment, the composition of the second aspect comprises lipid nanoparticles (LNPs), which have a molar ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e. proportion (mol %) of cationic lipid (preferably lipid III-3), DSPC, cholesterol and PEG-lipid ((preferably PEG-lipid of Formula (IVa) with n=49); solubilized in ethanol), wherein the lipid nanoparticles comprise at least one RNA encoding F (F0, F-del, F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0, F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1_mut3, F_DSCav1_mut4, F-del_DSCav1_mut4, F_DSCav1_mut5, F-del_DSCav1_mut5, F_DSCav1_mut6, F-del_DSCav1_mut6, F_DSCav1_mut7, F-del_DSCav1_mut7, F_DSCav1_mut8, or F-del_DSCav1_mut8), and at least one RNA encoding M, N, M2-1, or P.

The total amount of RNA in the lipid nanoparticles may vary and is defined depending on the e.g. RNA to total lipid w/w ratio. In one embodiment of the invention the artificial RNA to total lipid ratio is less than 0.06 w/w, preferably between 0.03 w/w and 0.04 w/w.

In various embodiments, the LNP as defined herein have a mean diameter of from about 50 nm to about 200 nm, from about 60 nm to about 200 nm, from about 70 nm to about 200 nm, from about 80 nm to about 200 nm, from about 90 nm to about 200 nm, from about 90 nm to about 190 nm, from about 90 nm to about 180 nm, from about 90 nm to about 170 nm, from about 90 nm to about 160 nm, from about 90 nm to about 150 nm, from about 90 nm to about 140 nm, from about 90 nm to about 130 nm, from about 90 nm to about 120 nm, from about 90 nm to about 100 nm, from about 70 nm to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm and are substantially non-toxic. As used herein, the mean diameter may be represented by the z-average as determined by dynamic light scattering as commonly known in the art.

In another preferred embodiment of the invention the lipid nanoparticles have a hydrodynamic diameter in the range from about 50 nm to about 300 nm, or from about 60 nm to about 250 nm, from about 60 nm to about 150 nm, or from about 60 nm to about 120 nm, respectively.

According to further embodiments, the composition of the second aspect may comprise at least one adjuvant. Suitably, the adjuvant is preferably added to enhance the immunostimulatory properties of the composition.

The term “adjuvant” as used herein will be recognized and understood by the person of ordinary skill in the art, and is for example intended to refer to a pharmacological and/or immunological agent that may modify, e.g. enhance, the effect of other agents (herein: the effect of the artificial nucleic acid of the invention) or that may be suitable to support administration and delivery of the composition. The term “adjuvant” refers to a broad spectrum of substances. Typically, these substances are able to increase the immunogenicity of antigens. For example, adjuvants may be recognized by the innate immune systems and, e.g., may elicit an innate immune response (that is, a non-specific immune response). “Adjuvants” typically do not elicit an adaptive immune response. In the context of the invention, adjuvants may enhance the effect of the antigenic peptide or protein provided by the artificial nucleic acid as defined herein or the polyprotein as defined herein.

In that context, the at least one adjuvant may be selected from any adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an immune response in a subject, e.g. in a human subject.

Accordingly, the composition of the second aspect may comprise at least one adjuvant, wherein the at least one adjuvant may be suitably selected from any adjuvant provided in of published PCT application WO2016/203025.

Adjuvants disclosed in any of the claims 2 to 17 of WO2016/203025, preferably adjuvants disclosed in claim 17 of WO2016/203025 are particularly suitable, the specific content relating thereto herewith incorporated by reference.

The composition of the second aspect may comprise, besides the components specified herein, at least one further component which may be selected from the group consisting of further antigens (e.g. in the form of a peptide or protein) or further antigen-encoding nucleic acids; a further immunotherapeutic agent; one or more auxiliary substances (cytokines, such as monokines, lymphokines, interleukins or chemokines); or any further compound, which is known to be immune stimulating due to its binding affinity (as ligands) to human Toll-like receptors; and/or an adjuvant nucleic acid, preferably an immunostimulatory RNA (isRNA), e.g. CpG-RNA etc.

Polypeptide and Composition Comprising the Polypeptide

In a third aspect, the present invention provides a polypeptide, preferably an antigenic polypeptide, wherein the polypeptide comprises an amino acid sequence or a fragment thereof encoded by the artificial RNA of the first aspect.

In embodiments, the polypeptide has an amino acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106, 19475 or a variant of any of these polypeptides.

In preferred embodiments, the polypeptide has an amino acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1267, 2005, 2743, 3481, 4219, 4957, 5695, 6433, 7171, 7909, 12833, 14309, 15047, 15785, 13571, 16523, 17261, 17999, 18737, 19475 or a variant of any of these polypeptides.

In a fourth aspect, the invention relates to an immunogenic composition, comprising the polypeptide of the third aspect. In preferred embodiments, the composition of the fourth aspect may additionally comprise at least one pharmaceutically acceptable carrier as defined herein. In preferred embodiments, the composition of the fourth aspect may additionally comprise at least one artificial RNA of the first aspect and, optionally, at least one further artificial RNA of the second aspect, or an RNA composition of the second aspect.

Notably, embodiments relating to the composition of the second aspect or the vaccine of the fifth aspect may likewise be read on and be understood as suitable embodiments of the composition of the fourth aspect.

Vaccine:

In a fifth aspect, the present invention provides a vaccine wherein the vaccine comprises the RNA of the first aspect, and, optionally, the composition of the second aspect, the polypeptide of the third aspect, or the composition of the fourth aspect.

Notably, embodiments relating to the composition of the second aspect or the composition of the fourth aspect may likewise be read on and be understood as suitable embodiments of the vaccine of the fifth aspect. Also, embodiments relating to the vaccine of the fifth aspect may likewise be read on and be understood as suitable embodiments of the composition of the second aspect (comprising the RNA of the first aspect and, optionally, the further artificial RNA of the second aspect) or the composition of the fourth aspect (comprising the polypeptide of the third aspect).

The term “vaccine” will be recognized and understood by the person of ordinary skill in the art, and is for example intended to be a prophylactic or therapeutic material providing at least one epitope or antigen, preferably an immunogen. In the context of the invention the antigen or antigenic function is provided by the inventive RNA of the first aspect (said RNA comprising a coding sequence encoding a antigenic peptide or protein derived from RSV F protein), the composition of the second aspect (comprising the RNA of the first aspect and, optionally, the further artificial RNA of the second aspect), the polypeptide of the third aspect, or the composition of the fourth aspect (comprising said polypeptide).

In preferred embodiments of the fifth aspect, the vaccine comprises the RNA of the first aspect, the composition of the second aspect (comprising the RNA of the first aspect and, optionally, the further artificial RNA of the second aspect), the polypeptide of the third aspect, or the composition of the fourth aspect wherein said RNA, said composition of the second aspect, said polypeptide, or said composition of the fourth aspect (comprising said polypeptide) elicits an adaptive immune response.

In particularly preferred embodiments, the vaccine comprises the RNA of the first aspect or the composition of the second aspect wherein said RNA or said composition elicits an adaptive immune response, preferably an adaptive immune response against RSV.

In particularly preferred embodiments, the vaccine comprises the RNA of the first aspect or the composition of the second aspect wherein said RNA or said composition induces a T cell immune response, According to a preferred embodiment of the fifth aspect, the vaccine as defined herein may further comprise a pharmaceutically acceptable carrier and optionally at least one adjuvant as specified in the context of the second aspect.

Suitable adjuvants in that context may be selected from adjuvants disclosed in claim 17 of WO2016/203025.

In a preferred embodiment, the vaccine is a monovalent vaccine.

In embodiments the vaccine is a polyvalent vaccine comprising a plurality or at least more than one of the artificial RNAs as defined in the context of the first aspect, and, optionally, at least more than one of the further artificial RNAs as defined in the context of the second aspect. Embodiments relating to a polyvalent composition as disclosed in the context of the second aspect may likewise be read on and be understood as suitable embodiments of the polyvalent vaccine of the fifth aspect.

The vaccine of the fifth aspect typically comprises a safe and effective amount of the RNA of the first aspect and, optionally, the further RNA of the second aspect. As used herein, “safe and effective amount” means an amount of the RNA that is sufficient to significantly induce a positive modification of a disease or disorder related to an infection with a RSV. At the same time, a “safe and effective amount” is small enough to avoid serious side-effects. In relation to the vaccine or composition of the present invention, the expression “safe and effective amount” preferably means an amount of the RNA that is suitable for stimulating the adaptive immune system in such a manner that no excessive or damaging immune reactions are achieved but, preferably, also no such immune reactions below a measurable level.

A “safe and effective amount” of the RNA of the composition or vaccine as defined above will furthermore vary in connection with the particular condition to be treated and also with the age and physical condition of the patient to be treated, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable carrier used, and similar factors, within the knowledge and experience of the accompanying medical doctor. Moreover, the “safe and effective amount” of the RNA, the composition, the vaccine may depend from application route (intradermal, intramuscular), application device (jet injection, needle injection, microneedle patch) and/or complexation (protamine complexation or LNP encapsulation). Moreover, the “safe and effective amount” of the artificial RNA, the composition, the vaccine may depend from the condition of the treated subject (infant, pregnant women, immunocompromised human subject etc.). Accordingly, the suitable “safe and effective amount” has to be adapted accordingly and will be chosen and defined by the skilled person.

The vaccine can be used according to the invention for human medical purposes and also for veterinary medical purposes (mammals, vertebrates, avian species), as a pharmaceutical composition, or as a vaccine.

In a preferred embodiment, the RNA, the composition (comprising said RNA and, optionally, a further RNA), the polypeptide, the composition (comprising said polypeptide), or the vaccine is provided in lyophilized form (using e.g. lyophilisation methods as described in WO2016/165831, WO2011/069586, WO2016/184575 or WO2016/184576). Preferably, the lyophilized RNA, the lyophilized composition, the lyophilized polypeptide, the lyophilized composition comprising the polypeptide, or the lyophilized vaccine is reconstituted in a suitable buffer, advantageously based on an aqueous carrier, prior to administration, e.g. Ringer-Lactate solution or a phosphate buffer solution.

Accordingly, the pharmaceutically acceptable carrier as used herein preferably includes the liquid or non-liquid basis of the inventive vaccine. If the inventive vaccine is provided in liquid form, the carrier will be water, typically pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g. phosphate, citrate etc. buffered solutions. Preferably, Ringer-Lactate solution is used as a liquid basis for the vaccine or the composition according to the invention as described in WO2006/122828, the disclosure relating to suitable buffered solutions incorporated herewith by reference.

The choice of a pharmaceutically acceptable carrier as defined herein is determined, in principle, by the manner, in which the pharmaceutical composition(s) or vaccine according to the invention is administered. The vaccine can be administered, for example, systemically or locally. Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intra-arterial, intradermal and intraperitoneal injections and/or intranasal administration routes. Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, intraarticular and sublingual injections. More preferably, composition or vaccines according to the present invention may be administered by an intradermal, subcutaneous, or intramuscular route, preferably by injection, which may be needle-free and/or needle injection. Compositions/vaccines are therefore preferably formulated in liquid or solid form. The suitable amount of the vaccine or composition according to the invention to be administered can be determined by routine experiments, e.g. by using animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models. Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices. Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams, gels and the like. If the inventive compositionor vaccine is to be administered perorally, tablets, capsules and the like are the preferred unit dose form. The pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art.

The inventive vaccine or composition as defined herein can additionally contain one or more auxiliary substances as defined above in order to further increase the immunogenicity. A synergistic action of the nucleic acid contained in the inventive composition and of an auxiliary substance, which may be optionally be co-formulated (or separately formulated) with the inventive vaccine or composition as described above, is preferably achieved thereby. Such immunogenicity increasing agents or compounds may be provided separately (not co-formulated with the inventive vaccine or composition) and administered individually.

Further additives which may be included in the inventive vaccine or composition are emulsifiers, such as for example, Tween; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; preservatives.

Kit or Kit of Parts, Application, Medical Uses, Method of Treatment:

In a sixth aspect, the present invention provides a kit or kit of parts, wherein the kit or kit of parts comprises the RNA of the first aspect, the composition of the second aspect (comprising said RNA), the polypeptide of the third aspect, the composition of the fourth aspect (comprising said polypeptide), and/or the vaccine of the fifth aspect, optionally comprising a liquid vehicle for solubilising, and optionally technical instructions providing information on administration and dosage of the components.

In a preferred embodiment, the kit of the sixth aspect comprises at least the following components

-   a) at least one RNA of the first aspect, preferably encoding at     least one antigenic peptide or protein derived from a RSV fusion (F)     protein (RNA sequences preferably selected from Table 5 or 6),     wherein said artificial RNA is preferably complexed with one or more     lipids thereby forming lipid nanoparticles (LNP); and -   b) at least one, two, or three further artificial RNA encoding an     antigenic peptide or protein derived from RSV selected from M, N,     M2-1, or P (RNA sequences preferably selected from Table 7A),     wherein said further RNA is preferably complexed with one or more     lipids thereby forming lipid nanoparticles (LNP), wherein     components a) and b) are provided as separate entities or as a     single entity.

In a preferred embodiment, the kit of the sixth aspect comprises at least the following components

-   a) at least one RNA of the first aspect, preferably encoding at     least one antigenic peptide or protein derived from a RSV fusion (F)     protein (RNA sequences preferably selected from Table 5 or 6),     wherein said artificial RNA is preferably complexed with one or more     lipids thereby forming lipid nanoparticles (LNP); and -   b) at least one further artificial RNA encoding an antigenic peptide     or protein derived from RSV selected from M (RNA sequences     preferably selected from Table 7A, Column A), wherein said further     RNA is preferably complexed with one or more lipids thereby forming     lipid nanoparticles (LNP); and -   c) at least one further artificial RNA encoding an antigenic peptide     or protein derived from RSV selected from P (RNA sequences     preferably selected from Table 7A, Column C), wherein said further     RNA is preferably complexed with one or more lipids thereby forming     lipid nanoparticles (LNP),

wherein components a), b) and c) are provided as separate entities or as a single entity.

In a preferred embodiment, the kit of the sixth aspect comprises at least the following components

-   a) at least one RNA of the first aspect, preferably encoding at     least one antigenic peptide or protein derived from a RSV fusion (F)     protein (RNA sequences preferably selected from Table 5 or 6),     wherein said artificial RNA is preferably complexed with one or more     lipids thereby forming lipid nanoparticles (LNP); and -   b) at least one further artificial RNA encoding an antigenic peptide     or protein derived from RSV selected from M (RNA sequences     preferably selected from Table 7A, Column A), wherein said further     RNA is preferably complexed with one or more lipids thereby forming     lipid nanoparticles (LNP); and -   c) at least one further artificial RNA encoding an antigenic peptide     or protein derived from RSV selected from P (RNA sequences     preferably selected from Table 7A, Column C), wherein said further     RNA is preferably complexed with one or more lipids thereby forming     lipid nanoparticles (LNP); and -   d) at least one further artificial RNA encoding an antigenic peptide     or protein derived from RSV selected from P (RNA sequences     preferably selected from Table 7A, Column B), wherein said further     RNA is preferably complexed with one or more lipids thereby forming     lipid nanoparticles (LNP),

wherein components a), b), c) and d) are provided as separate entities or as a single entity.

The kit may further comprise additional components as described in the context of the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, and/or the vaccine of the fifth aspect.

The technical instructions of said kit may contain information about administration and dosage and patient groups. Such kits, preferably kits of parts, may be applied e.g. for any of the applications or uses mentioned herein, preferably for the use of the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, or the vaccine of the fifth aspect, for the treatment or prophylaxis of an infection or diseases caused by RSV or disorders related thereto. Preferably, the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, or the vaccine of the fifth aspect is provided in a separate part of the kit, wherein the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, or the vaccine of the fifth aspect is preferably lyophilised. The kit may further contain as a part a vehicle (e.g. buffer solution) for solubilising the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition fourth aspect, or the vaccine of the fifth aspect.

In preferred embodiments, the kit or kit of parts as defined herein comprises Ringer lactate solution.

Any of the above kits may be used in a treatment or prophylaxis as defined herein. More preferably, any of the above kits may be used as a vaccine, preferably a vaccine against infections caused by RSV as defined herein.

Medical Use:

In a further aspect, the present invention relates to the first medical use of the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect.

Accordingly, the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect is for use as a medicament.

The present invention furthermore provides several applications and uses of the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect.

In particular, said RNA, composition (comprising said RNA), polypeptide, composition (comprising said polypeptide), vaccine, or the kit or kit of parts may be used for human medical purposes and also for veterinary medical purposes, preferably for human medical purposes.

In particular, said RNA, composition (comprising said RNA), polypeptide, composition (comprising said polypeptide), vaccine, or the kit or kit of parts may is for use as a medicament for human medical purposes, wherein said RNA, composition (comprising said RNA), polypeptide, composition (comprising said polypeptide), vaccine, or the kit or kit of parts may be particularly suitable for young infants, newborns, immunocompromised recipients, as well as pregnant and breast-feeding women and elderly people.

In yet another aspect, the present invention relates to the second medical use of the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect.

Accordingly, the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect is for use in the treatment or prophylaxis of an infection with a pathogen (e.g. a virus), in particular with Respiratory syncytial virus (RSV), or a disorder related to such an infection.

In particular, the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect may be used in the treatment or prophylaxis of an infection with a virus, in particular with RSV, or a disorder related to such an infection for human and also for veterinary medical purposes, preferably for human medical purposes.

In particular, said RNA, composition (comprising said RNA), polypeptide, composition (comprising said polypeptide), vaccine, or the kit or kit of parts may be particularly suitable for young infants, newborns, immunocompromised recipients, as well as pregnant and breast-feeding women and elderly people for use in the treatment or prophylaxis of an infection with RSV.

As used herein, “a disorder related to a RSV infection” may preferably comprise a typical symptom or a complication of an RSV infection.

Particularly, the artificial RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect may be used in a method of prophylactic (pre-exposure prophylaxis or post-exposure prophylaxis) and/or therapeutic treatment of infections caused by RSV.

The composition or the vaccine as defined herein may preferably be administered locally. In particular, composition or vaccines may be administered by an intradermal, subcutaneous, intranasal, or intramuscular route. Inventive compositions or vaccines of the invention are therefore preferably formulated in liquid (or sometimes in solid) form. In embodiments, the inventive vaccine may be administered by conventional needle injection or needle-free jet injection. Preferred in that context is the RNA, the composition, the vaccine is administered by intramuscular needle injection.

The term “jet injection”, as used herein, refers to a needle-free injection method, wherein a fluid (vaccine, composition of the invention) containing e.g. at least one RNA of the first aspect is forced through an orifice, thus generating an ultra-fine liquid stream of high pressure that is capable of penetrating mammalian skin and, depending on the injection settings, subcutaneous tissue or muscle tissue. In principle, the liquid stream perforates the skin, through which the liquid stream is pushed into the target tissue. Preferably, jet injection is used for intradermal, subcutaneous or intramuscular injection of the RNA, the compositions, the vaccines disclosed herein.

In embodiments, the RNA as comprised in a composition or vaccine as defined herein is provided in an amount of about 100 ng to about 500 μg, in an amount of about 1 μg to about 200 μg, in an amount of about 1 μg to about 100 μg, in an amount of about 5 μg to about 100 μg, preferably in an amount of about 10 μg to about 50 μg, specifically, in an amount of about 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg or 100 μg.

Depending from application route (intradermal, intramuscular, intranasal), application device (jet injection, needle injection, microneedle patch) and/or complexation (preferably LNP encapsulation) the suitable amount has to be adapted accordingly and will be chosen and defined by the skilled person.

The immunization protocol for the treatment or prophylaxis of an infection as defined herein, i.e. the immunization of a subject against a RSV, typically comprises a series of single doses or dosages of the composition or the vaccine. A single dosage, as used herein, refers to the initial/first dose, a second dose or any further doses, respectively, which are preferably administered in order to “boost” the immune reaction.

In one embodiment, the immunization protocol for the treatment or prophylaxis of an infection as defined herein, i.e. the immunization of a subject against a RSV, comprises one single doses of the composition or the vaccine.

In preferred embodiments, the immunization protocol for the prophylaxis of an infection with RSV comprises at least one single dose of the composition or the vaccine as defined herein, wherein said at least one single dose is administered to a pregnant women for maternal immunization, thereby achieving immunization of the unborn child and/or wherein said at least one single dose is administered to a breast-feeding women for thereby achieving passive immunization of the breast-fed child.

The treatment or prophylaxis as defined above may comprise the administration of a further active pharmaceutical ingredient. In the case of the inventive vaccine or composition, which is based on the artificial RNA of the first aspect, a polypeptide, preferably of the third aspect may be co-administered as a further active pharmaceutical ingredient.

For example, at least one RSV protein or peptide as described herein, or a fragment or variant thereof, may be co-administered in order to induce or enhance an immune response. Further, two distinct artificial RNAs of the first aspect and, optionally further RNAs of the second aspect may be administered at different time points, preferably in a prime-boost scenario, e.g. using a composition comprising at least one RSV polypeptide as prime vaccination and a composition/vaccine comprising at least one artificial RNA of the first aspect as boost vaccination.

Suitably, the treatment or prophylaxis as defined above comprises the administration of a further active pharmaceutical ingredient, wherein the further active pharmaceutical ingredient may be an immunotherapeutic agent that can be selected from immunoglobulins, preferably IgGs, monoclonal or polyclonal antibodies, polyclonal serum or sera, etc., most preferably immunoglobulins directed against a RSV protein or peptide as defined herein. Preferably, such a further immunotherapeutic agent may be provided as a peptide/protein or may be encoded by a nucleic acid, preferably by a DNA or an RNA, more preferably an mRNA. Such an immunotherapeutic agent allows providing passive vaccination additional to active vaccination triggered by the inventive artificial RNA or by the inventive polypeptide.

Method of Treatment and Use, Diagnostic Method and Use:

In another aspect, the present invention relates to a method of treating or preventing a disorder.

In preferred embodiments, the present invention relates to a method of treating or preventing a disorder, wherein the method comprises applying or administering to a subject in need thereof the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect.

In preferred embodiments, the disorder is an infection with Respiratory syncytial virus (RSV), or a disorder related to such an infection.

In preferred embodiments, the present invention relates to a method of treating or preventing a disorder, wherein the method comprises applying or administering to a subject in need thereof the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect, wherein the subject in need is preferably a mammalian subject. In particularly preferred embodiments, the mammalian subject is a human subject, particularly an infant, a newborn, a pregnant women, a breast-feeding woman, an elderly, or an immunocompromised human subject.

In particular, such a method may preferably comprise the steps of:

-   a) providing the RNA of the first aspect, the composition of the     second aspect, the polypeptide of the third aspect, the composition     of the fourth aspect, the vaccine of the fifth aspect, or the kit or     kit of parts of the sixth aspect; -   b) applying or administering said RNA of the first aspect,     composition of the second aspect, polypeptide of the third aspect,     composition of the fourth aspect, vaccine of the fifth aspect, or     kit or kit of parts of the sixth aspect to a tissue or an organism; -   c) optionally, administering immunoglobulin (IgGs) against a RSV; -   d) optionally, administering a further substance (adjuvant,     auxiliary substance, further antigen).

According to a further aspect, the present invention also provides a method for expression of at least one polypeptide comprising at least one peptide or protein derived from a RSV, or a fragment or variant thereof, wherein the method preferably comprises the following steps:

-   a) providing the RNA of the first aspect or the composition of the     second aspect; and -   b) applying or administering said RNA or composition to an     expression system (cells), a tissue, an organism.

The method may be applied for laboratory, for research, for diagnostic, for commercial production of peptides or proteins and/or for therapeutic purposes. The method may furthermore be carried out in the context of the treatment of a specific disease, particularly in the treatment of infectious diseases, particularly RSV infections.

Likewise, according to another aspect, the present invention also provides the use of the artificial RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect preferably for diagnostic or therapeutic purposes, e.g. for expression of an encoded RSV antigenic peptide or protein, e.g. by applying or administering said RNA, composition comprising said RNA, vaccine comprising said RNA, e.g. to a cell-free expression system, a cell (e.g. an expression host cell or a somatic cell), a tissue or an organism. In specific embodiments, applying or administering said RNA, composition comprising said RNA, vaccine comprising said RNA to a tissue or an organism is followed by e.g. a step of obtaining induced RSV F antibodies e.g. RSV F specific (monoclonal) antibodies.

The use may be applied for a (diagnostic) laboratory, for research, for diagnostics, for commercial production of peptides, proteins, or RSV antibodies and/or for therapeutic purposes. The use may be carried out in vitro, in vivo or ex vivo. The use may furthermore be carried out in the context of the treatment of a specific disease, particularly in the treatment of an RSV infection or a related disorder.

In a particularly preferred embodiment, the invention provides the RNA of the first aspect, the composition of the second aspect, the polypeptide of the third aspect, the composition of the fourth aspect, the vaccine of the fifth aspect, or the kit or kit of parts of the sixth aspect for use as a medicament, for use in treatment or prophylaxis, preferably treatment or prophylaxis of an RSV infection or a related disorder, or for use as a vaccine.

BRIEF DESCRIPTION OF LISTS AND TABLES

List 1: Suitable RSV virus strains

List 2: NCBI Protein Accession numbers of suitable RSV fusion (F) proteins

Table 1: Preferred RSV F protein antigen designs

Table 2: Human codon usage table with frequencies indicated for each amino acid

Table 3A: Preferred coding sequences encoding RSV F (columns A-J), derived from HRSV(A2)

Table 3B: Preferred coding sequences encoding RSV F (columns A-J), derived from HRSV(Memphis-37)

Table 4A: Preferred coding sequences encoding RSV F (columns K-V), derived from HRSV(A2)

Table 4B: Preferred coding sequences encoding RSV F (columns K-V), derived from HRSV(Memphis-37)

Table 5A: Preferred mRNA constructs encoding RSV F (columns A-J), derived from HRSV(A2)

Table 5B: Preferred mRNA constructs encoding RSV F (columns A-J), derived from HRSV(Memphis-37)

Table 6A: Preferred mRNA constructs encoding RSV F (column K-V), derived from HRSV(A2)

Table 6B: Preferred mRNA constructs encoding RSV F (column K-V), derived from HRSV(Memphis-37)

Table 7A: Preferred further coding sequences and mRNA constructs of the composition or vaccine

Table 7B: Suitable combinations of RNA constructs

Table 8: Representative lipid compounds derived from formula (III)

Table 9: mRNA constructs used in the present examples (see Examples section)

Table 10: Animal groups and vaccination schedule of Example 2 (see Examples section)

Table 11: Animal groups and vaccination schedule of Example 3 (see Examples section)

Table 12: mRNA constructs with different UTR combinations of Example 4 (see Examples section)

Table 13: Animal groups and vaccination schedule of Example 5 (see Examples section)

Table 14: Animal groups and vaccination schedule of Example 6 (see Examples section)

Table 15: Animal groups and vaccination schedule of Example 7 (see Examples section)

Table 15: Animal groups and vaccination schedule of Example 7 (see Examples section)

Table 16: Overview of mRNA constructs used in Example 8 (see Examples section)

Table 17: Animal groups and vaccination schedule of Example 9 (see Examples section)

Table 18: Animal groups and vaccination schedule of Example 10 (see Examples section)

Table 19: Overview of mRNA constructs used in Example 11 (see Examples section)

Table 20: Animal groups and vaccination schedule of Example 12 (see Examples section)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that LNP-formulated mRNA encoding RSV F-protein (F-del_DSCav1) induces high virus neutralization titers (VNTs) in serum of cotton rats after i.m. and i.d. vaccinations. Vaccination with protamine-formulated mRNA induces only weak responses. Vaccination schedule see Table 10. Further details are provided in Example 2.

FIG. 2 shows that LNP-formulated mRNA encoding RSV F-protein (F-del_DSCav1) reduces RSV infection of the lung in cotton rats after i.m. and i.d. vaccination. Vaccination schedule see Table 10. Further details are provided in Example 2.

FIG. 3 shows the results of a lung histopathology analysis from the RSV cotton rat challenge study described in Example 2. Animals vaccinated with LNP-formulated mRNA do not show increased lung histopathology/enhanced inflammation after viral challenge in contrast to the vaccination with formalin-inactivated RSV virus vaccine. Vaccination schedule see Table 10. Further details are provided in Example 2.

FIG. 4 shows that LNP-formulated mRNA encoding RSV F-protein (F0, F-del, and F-del_DSCav1) induces high (virus neutralization titers) VNTs in the serum of cotton rats after two i.m. vaccinations. VNTs determined performed on day 48. Vaccination schedule see Table 11. Further details are provided in Example 3.

FIG. 5 shows that LNP-formulated mRNA encoding RSV F-protein (F0, F-del, and F-del_DSCav1) induces specific humoral immune responses in cotton rats against the RSV-F protein. The experiment was performed as described in Example 3 and antibody total IgG titers were determined by ELISA. RSV-F (formulated in LNP) and RSV-F-del (formulated in LNP) vaccines induce higher titers of RSV-F specific IgGs already after prime vaccination (day 28) than live virus. FIG. 5a : Antibody titers determined on day 49; FIGS. 5b and 5c : time-dependent IgG titers in serum with 10 μg mRNA (FIG. 5b ) or 100 μg mRNA (FIG. 5c ) encoding RSV F-protein (F, F-del, and F-del_DSCav1). Vaccination schedule see Table 11. Further details are provided in Example 3.

FIG. 6 shows that LNP-formulated mRNA encoding RSV F-protein (F0, F-del, and F-del_DSCav1) reduces RSV infection of the lung in cotton rats after i.m. vaccination. The experiment was performed as described in Example 3. All animal groups vaccinated with mRNA vaccines showed virus titers below the level of detection demonstrating protection of vaccinated cotton rats in terms of viral lung titers. In comparison to the mRNA vaccines the vaccine based on formalin-inactivated virus was not able to prevent virus titers in the lung. Vaccination schedule see Table 11. Further details are provided in Example 3.

FIG. 7 shows that LNP-formulated mRNA encoding RSV F-protein (F0, F-del, and F-del_DSCav1) reduces RSV titers in the nose of cotton rats after i.m. vaccination. The experiment was performed as described in Example 3. All animal groups vaccinated with mRNA vaccines show strongly reduced viral titers in the nasal tissue in viral challenge infection experiments. In comparison to the mRNA vaccines the vaccine based on formalin-inactivated virus was not able to reduce nasal virus titers. Vaccination schedule see Table 11. Further details are provided in Example 3.

FIG. 8 shows the results of a lung histopathology analysis from the RSV cotton rat challenge study described in Example 3. Animals vaccinated with LNP-formulated mRNA do not show increased lung histopathology/enhanced inflammation after viral challenge in contrast to the vaccination with formalin-inactivated RSV virus vaccine. Vaccination schedule see Table 11. Further details are provided in Example 3.

FIG. 9 shows that UTR combinations according to the invention increase the expression of RSV F-Protein in vitro. HEK 293T cells were transfected with different Lipofectamine-formulated mRNA constructs, which all contain the same coding sequence encoding pre-fusion stabilized truncated RSV-F (F-del_DSCav1) but use different combinations of 5′ and 3′-UTRs (see Table 12). RSV-F expression was analyzed by flow cytometry. Values show % of the detected RSV-F signal. Values normalized to 100% according to the expression of the reference mRNA construct (UTR combination RPL32/ALB7). Water for injection (WFI) serves as a control. N=2. Further details are provided in Example 4.

FIG. 10 shows that the used mRNA constructs encoding RSV F-protein (FIG. 10a : F0, F-del, F0_DSCav1, F-del_DSCav1, F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1_mut3, FIG. 10b : F0, F-del, F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0, F_DSCav1_mut4, F-del_DSCav1_mut4, F_DSCav1_mut5, F-del_DSCav1_mut5) led to a detectable intracellular RSV F protein expression as well as to a detectable protein expression at the cell surface. Further details are provided in Table 16 and Example 8.

FIG. 11 shows that all tested LNP-formulated mRNA constructs encoding different RSV F proteins (F0, F0_DSCav1, F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del, F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) induce humoral immune responses in cotton rats against the RSV-F protein. Antibody total IgG titers were determined by ELISA. Vaccination schedule see Table 17. Further details are provided in Example 9.

FIG. 12 shows that all LNP-formulated RSV-F (F0, F0_DSCav1, F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del, F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) mRNA vaccines induced the formation of RSV specific functional antibodies in cotton rats as shown by high virus neutralizing antibody titers. Vaccination schedule see Table 17. Further details are provided in Example 9.

FIG. 13 shows the lung histopathology analysis from the RSV cotton rat challenge study. None of the mRNA vaccinated groups displayed enhanced lung pathology as it is the case for the group that was vaccinated using the formalin-inactivated RSV vaccine Vaccination schedule see Table 17. Further details are provided in Example 9.

FIG. 14 shows the results of the analysis of lung viral titers (FIG. 14a ) and of nose viral titers (FIG. 14b ) in cotton rats challenged with RSV virus. Vaccination schedule see Table 17. Further details are provided in Example 9.

FIG. 15 shows that all tested LNP-formulated mRNA constructs encoding different RSV F proteins (F-del, F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) induce humoral immune responses in cotton rats against the RSV-F protein. Antibody total IgG titers were determined by ELISA. Vaccination schedule see Table 18. Further details are provided in Example 10.

FIG. 16 shows that all LNP-formulated RSV-F (F-del, F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) mRNA vaccines induced the formation of RSV specific functional antibodies in cotton rats as shown by high virus neutralizing antibody titers. The LNP-formulated constructs for pre-fusion stabilization induce higher or comparable responses than F-del. Vaccination schedule see Table 18. Further details are provided in Example 10.

FIG. 17 shows the lung histopathology analysis from the RSV cotton rat challenge study. None of the mRNA vaccinated groups displayed enhanced lung pathology as it is the case for the group that was vaccinated using the formalin-inactivated RSV vaccine Vaccination schedule see Table 18. Further details are provided in Example 10.

FIG. 18 shows the results of the analysis of lung viral titers (FIG. 18a ) and of nose viral titers (FIG. 18b ) in cotton rats challenged with RSV virus. Vaccination schedule see Table 18. Further details are provided in Example 10.

FIG. 19 shows that the used mRNA constructs coding for RSV matrix protein M, phosphoprotein P, nucleoprotein N and matrix protein M2-1 protein led to a detectable protein expression using a rabbit reticulocyte lysate system. Further details are provided in Table 19 and Example 11.

FIG. 20 shows the results of the immunogenicity study in mice (ELISA). The humoral immune responses can be seen in FIG. 20a : anti-RSV F IgG and in FIG. 20b : anti-RSV F IgG2a. All groups induced humoral immune responses. In general, the IgG2a titers are 10 times higher than the IgG1 titers, indicating a predominant Th1 response. Vaccination schedule see Table 20. Further details are provided in Example 12.

FIG. 21 shows that specific antigen IgGs were detected in sera of immunized mice indicating that the applied mRNA constructs are suitable to induce specific humoral immune responses. Vaccination schedule see Table 20. Further details are provided in Example 12.

FIG. 22 shows that all LNP-formulated mRNA vaccines (F, M+F, P+F, N+F, M2-1+F) induced the formation of RSV specific functional antibodies in mice as shown by high virus neutralizing antibody titers. Vaccination schedule see Table 20. Further details are provided in Example 12.

FIG. 23 shows that all vaccines containing RSV F, RSV M2-1, or both surprisingly induced a tissue resident memory T cell (TRM) response in the lung upon intramuscular immunization (FIG. 23a ). The splenocyte analysis revealed that an immunization with RSV F and especially RSV M2-1 lead to an increase of antigen-specific CD8+ and CD4+ T cells secreting IFN-γ and TNF indicating the induction of a systemic T cell response in addition to a site-specific response (FIG. 23b and FIG. 23c ).

EXAMPLES

In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.

Example 1: Preparation of DNA and mRNA Constructs and Compositions for In Vitro and In Vivo Experiments

The present Example provides methods of obtaining the artificial RNA of the invention as well as methods of generating a composition or a vaccine of the invention.

1.1. Preparation of DNA and mRNA constructs:

For the present examples, DNA sequences encoding different RSV F proteins (e.g. F0, F-del, F-del_DSCav1, F-del_DSCav1_mut5, etc.) were prepared and used for subsequent RNA in vitro transcription reactions. Said DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a G/C optimized coding sequence (e.g., “cds opt1”) for stabilization. Sequences were introduced into a pUC19 derived vector to comprise stabilizing 3′-UTR sequences derived from a 3′-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1 and RPS9 and 5′-UTR sequences derived from a 5′-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, additionally comprising, a stretch of adenosines (e.g. 64A or A100), and a histone-stem-loop (hSL) structure, and optionally a stretch of 30 cytosines (e.g. C30) as listed in Table 9.

The obtained plasmid DNA constructs were transformed and propagated in bacteria using common protocols known in the art. Eventually, the plasmid DNA constructs were extracted, purified, and used for subsequent RNA in vitro transcription (see section 1.2.).

Alternatively, DNA plasmids prepared according to paragraph 1 are used as DNA template for PCR-based amplification. Eventually, the generated PCR products are purified and used for subsequent RNA in vitro transcription (see section 1.3.).

1.2. RNA In Vitro Transcription from Plasmid DNA Templates:

DNA plasmids prepared according to paragraph 1.1 were enzymatically linearized using EcoRI or SapI and used for DNA dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (e.g., m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG, or m7G(5′)ppp(5′)(2′OMeG)pG)) under suitable buffer conditions. The obtained mRNA constructs were purified using RP-HPLC (PureMessenger®, CureVac AG, Tubingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments. RNA for clinical development (see Example 6) is produced under current good manufacturing practice e.g. according to WO2016/180430, implementing various quality control steps on DNA and RNA level. The generated RNA sequences/constructs are provided in Table 9 with the encoded F protein and the respective UTR elements indicated therein. In addition to the information provided in Table 9, further information relating to specific mRNA construct SEQ-ID NOs may be derived from the information provided under <223> identifier provided in the ST.25 sequence listing.

Alternatively, EcoRI or SapI linearized DNA is used for DNA dependent RNA in vitro transcription using an RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N(1)-methylpseudouridine (m14) or pseudouridine (Ψ)) and cap analog (m7GpppG, m7G(5′)ppp(5′)(2′OMeA)pG, or m7G(5′)ppp(5′)(2′OMeG)pG) under suitable buffer conditions. The obtained m1Ψ or Ψ-modified mRNAs are purified using RP-HPLC (PureMessenger®, CureVac, Tobingen, Germany; WO2008/077592) and used for further experiments.

Some mRNA constructs are in vitro transcribed in the absence of a cap analog. The cap-structure (cap1) is added enzymatically using Capping enzymes as commonly known in the art. In short, in vitro transcribed mRNA is capped using an m7G capping kit with 2′-O-methyltransferase to obtain cap1-capped mRNA. Cap1-capped mRNA is purified using RP-HPLC (PureMessenger®, CureVac, Tubingen, Germany; WO2008/077592) and used for further experiments.

1.3. RNA In Vitro Transcription from PCR Amplified DNA Templates:

Purified PCR amplified DNA templates prepared according to paragraph 1.1 are transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and cap analog (m7GpppG) under suitable buffer conditions. Alternatively, PCR amplified DNA is transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a modified nucleotide mixture (ATP, GTP, CTP, N(1)-methylpseudouridine (m14)) and cap analog (m7GpppG) under suitable buffer conditions. Some mRNA constructs are in vitro transcribed in the absence of a cap analog and the cap-structure (cap1) is added enzymatically using capping enzymes as commonly known in the art e.g. using an m7G capping kit with 2′-O-methyltransferase. The obtained mRNAs are purified e.g. using RP-HPLC (PureMessenger®, CureVac AG, Tübingen, Germany; WO2008/077592) and used for in vitro and in vivo experiments.

TABLE 9 mRNA constructs used in the present examples RNA 5′-UTR/3′-UTR; 3′-terminal SEQ ID NO: SEQ ID NO: ID Antigen Name UTR Design Elements (3′-end) RNA Protein R6939/ F0 —/muag; i-3 A64-N5-C30-histoneSL-N5 475 68 R7003 R3737/ F0 RPL32/ALB7; i-2 A64-N5-C30-histoneSL-N5 466 68 R3938 R6940 F-del —/muag; i-3 A64-N5-C30-histoneSL-N5 890 483 R3738/ F-del RPL32/ALB7; i-2 A64-N5-C30-histoneSL-N5 881 483 R3939 R6808 F0_DSCav1 —/muag; i-3 A64-N5-C30-histoneSL-N5 1259 898 R5453 F-del_DSCav1 —/muag; i-3 A64-N5-C30-histoneSL-N5 1628 1267 R6122 F-del_DSCav1 RPL32/ALB7; i-2 A64-N5-C30-histoneSL-N5 1620 1267 R4745/ F-del_DSCav1 HSD17B4/ALB7; i-4 A64-N5-C30-histoneSL-N5 8278 1267 R5717 R6771 F_DSCav1_mut1 —/muag; i-3 A64-N5-C30-histoneSL-N5 1997 1636 R6774 F-del_DSCav1_mut1 —/muag; i-3 A64-N5-C30-histoneSL-N5 2366 2005 R6772 F_DSCav1_mut2 —/muag; i-3 A64-N5-C30-histoneSL-N5 2735 2374 R6773 F-del_DSCav1_mut2 —/muag; i-3 A64-N5-C30-histoneSL-N5 3104 2743 R6770 F_DSCav1_mut3 —/muag; i-3 A64-N5-C30-histoneSL-N5 3473 3112 R6775 F-del_DSCav1_mut3 —/muag; i-3 A64-N5-C30-histoneSL-N5 3842 3481 F-del_DSCav1 HSD17B4/PSMB3; a-1 A64-N5-C30-histoneSL-N5 1276 1267 F-del_DSCav1 Ndufa4/PSMB3; a-2 A64-N5-C30-histoneSL-N5 1284 1267 F-del_DSCav1 Slc7a3/PSMB3; a-3 A64-N5-C30-histoneSL-N5 1292 1267 F-del_DSCav1 Nosip/PSMB3; a-4 A64-N5-C30-histoneSL-N5 1300 1267 F-del_DSCav1 Mp68/PSMB3; a-5 A64-N5-C30-histoneSL-N5 1308 1267 F-del_DSCav1 Ubqln2/RPS9; b-1 A64-N5-C30-histoneSL-N5 1316 1267 F-del_DSCav1 ASAH1/RPS9; b-2 A64-N5-C30-histoneSL-N5 1324 1267 F-del_DSCav1 HSD17B4/RPS9; b-3 A64-N5-C30-histoneSL-N5 1332 1267 F-del_DSCav1 HSD17B4/CASP1; b-4 A64-N5-C30-histoneSL-N5 1340 1267 F-del_DSCav1 Nosip/COX6B1; b-5 A64-N5-C30-histoneSL-N5 1348 1267 F-del_DSCav1 Ndufa4/RPS9; c-1 A64-N5-C30-histoneSL-N5 1356 1267 F-del_DSCav1 Nosip/Ndufa1; c-2 A64-N5-C30-histoneSL-N5 1364 1267 F-del_DSCav1 Ndufa4/COX6B1; c-3 A64-N5-C30-histoneSL-N5 1372 1267 F-del_DSCav1 Ndufa4/Ndufa1; c-4 A64-N5-C30-histoneSL-N5 1380 1267 F-del_DSCav1 ATP5A1/PSMB3; c-5 A64-N5-C30-histoneSL-N5 1388 1267 F-del_DSCav1 Rpl31/PSMB3; d-1 A64-N5-C30-histoneSL-N5 1396 1267 F-del_DSCav1 ATP5A1/CASP1; d-2 A64-N5-C30-histoneSL-N5 1404 1267 F-del_DSCav1 Slc7a3/Gnas; d-3 A64-N5-C30-histoneSL-N5 1412 1267 F-del_DSCav1 HSD17B4/Ndufa1; d-4 A64-N5-C30-histoneSL-N5 1420 1267 F-del_DSCav1 Slc7a3/Ndufa1; d-5 A64-N5-C30-histoneSL-N5 1428 1267 F-del_DSCav1 TUBB4B/RPS9; e-1 A64-N5-C30-histoneSL-N5 1436 1267 F-del_DSCav1 Rpl31/RPS9; e-2 A64-N5-C30-histoneSL-N5 1444 1267 F-del_DSCav1 Mp68/RPS9; e-3 A64-N5-C30-histoneSL-N5 1452 1267 F-del_DSCav1 Nosip/RPS9; e-4 A64-N5-C30-histoneSL-N5 1460 1267 F-del_DSCav1 ATP5A1/RPS9; e-5 A64-N5-C30-histoneSL-N5 1468 1267 F-del_DSCav1 ATP5A1/COX6B1; e-6 A64-N5-C30-histoneSL-N5 1476 1267 F-del_DSCav1 ATP5A1/Gnas; f-1 A64-N5-C30-histoneSL-N5 1484 1267 F-del_DSCav1 ATP5A1/Ndufa1; f-2 A64-N5-C30-histoneSL-N5 1492 1267 F-del_DSCav1 HSD17B4/COX6B1; f-3 A64-N5-C30-histoneSL-N5 1500 1267 F-del_DSCav1 HSD17B4/Gnas; f-4 A64-N5-C30-histoneSL-N5 1508 1267 F-del_DSCav1 Mp68/COX6B1; f-5 A64-N5-C30-histoneSL-N5 1516 1267 F-del_DSCav1 Mp68/Ndufa1; g-1 A64-N5-C30-histoneSL-N5 1524 1267 F-del_DSCav1 Ndufa4/CASP1; g-2 A64-N5-C30-histoneSL-N5 1532 1267 F-del_DSCav1 Ndufa4/Gnas; g-3 A64-N5-C30-histoneSL-N5 1540 1267 F-del_DSCav1 Nosip/CASP1; g-4 A64-N5-C30-histoneSL-N5 1548 1267 F-del_DSCav1 Rpl31/CASP1; g-5 A64-N5-C30-histoneSL-N5 1556 1267 F-del_DSCav1 Rpl31/COX6B1; h-1 A64-N5-C30-histoneSL-N5 1564 1267 F-del_DSCav1 Rpl31/Gnas; h-2 A64-N5-C30-histoneSL-N5 1572 1267 F-del_DSCav1 Rpl31/Ndufa1; h-3 A64-N5-C30-histoneSL-N5 1580 1267 F-del_DSCav1 Slc7a3/CASP1; h-4 A64-N5-C30-histoneSL-N5 1588 1267 F-del_DSCav1 Slc7a3/COX6B1; h-5 A64-N5-C30-histoneSL-N5 1596 1267 F-del_DSCav1 Slc7a3/RPS9; i-1 A64-N5-C30-histoneSL-N5 1604 1267 F-del_DSCav1 RPL32(32L4)/ALB7; i-2 A64-N5-C30-histoneSL-N5 1612 1267 R7454 F_DSCav1_mut0 —/muag; i-3 A64-N5-C30-histoneSL-N5 4211 3850 R7455 F-del_DSCav1_mut0 —/muag; i-3 A64-N5-C30-histoneSL-N5 4580 4219 R7458 F_DSCav1_mut4 —/muag; i-3 A64-N5-C30-histoneSL-N5 4949 4588 R7459 F-del_DSCav1_mut4 —/muag; i-3 A64-N5-C30-histoneSL-N5 5318 4957 R7456 F_DSCav1_mut5 —/muag; i-3 A64-N5-C30-histoneSL-N5 5687 5326 R7457 F-del_DSCav1_mut5 —/muag; i-3 A64-N5-C30-histoneSL-N5 6056 5695 F_DSCav1_mut6 —/muag; i-3 A64-N5-C30-histoneSL-N5 6425 6064 F-del_DSCav1_mut6 —/muag; i-3 A64-N5-C30-histoneSL-N5 6794 6433 F_DSCav1_mut7 —/muag; i-3 A64-N5-C30-histoneSL-N5 7163 6802 F-del_DSCav1_mut7 —/muag; i-3 A64-N5-C30-histoneSL-N5 7532 7171 F_DSCav1_mut8 —/muag; i-3 A64-N5-C30-histoneSL-N5 7901 7540 F-del_DSCav1_mut8 —/muag; i-3 A64-N5-C30-histoneSL-N5 8270 7909 R7595 M —/muag; i-3 A64-N5-C30-histoneSL-N5 10046 9684 R7597 N —/muag; i-3 A64-N5-C30-histoneSL-N5 10496 10134 R7598 M2-1 —/muag; i-3 A64-N5-C30-histoneSL-N5 11545 11183 R7596 P —/muag; i-3 A64-N5-C30-histoneSL-N5 10999 10637

1.4. Preparation of an LNP Formulated mRNA Composition:

Lipid nanoparticles (LNP), cationic lipids, and polymer conjugated lipids (PEG-lipid) were prepared and tested essentially according to the general procedures described in WO2015/199952, WO2017/004143 and WO2017/075531, the full disclosures of which are incorporated herein by reference. LNP formulated mRNA was prepared using an ionizable amino lipid (cationic lipid), phospholipid, cholesterol and a PEGylated lipid. Briefly, cationic lipid compound of formula III-3, DSPC, cholesterol, and PEG-lipid of formula Va were solubilized in ethanol at a molar ratio (%) of approximately 50:10:38.5:1.5 or 47.4:10:40.9:1.7. LNPs comprising cationic lipid compound of formula III-3 and PEG-lipid compound of formula IVa were prepared at a ratio of mRNA to total Lipid of 0.03-0.04 w/w. The mRNA was diluted to 0.05 mg/mL to 0.2 mg/mL in 10 mM to 50 mM citrate buffer, pH4. Syringe pumps were used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 15 ml/min. The ethanol was then removed and the external buffer replaced with a PBS buffer comprising Sucrose by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 um pore sterile filter and the LNP-formulated mRNA composition was adjusted to about 1 mg/ml total mRNA. Lipid nanoparticle particle diameter size was 60-90 nm as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK). For other cationic lipid compounds mentioned in the present specification, the formulation process is essentially similar. The obtained LNP-formulated mRNA composition (1 mg/ml total mRNA) was diluted to the desired target concentration using Saline before in vivo application.

1.5. Preparation of a Protamine Complexed mRNA Composition:

mRNA constructs were complexed with protamine prior to use in in vivo immunization experiments. The mRNA formulation consisted of a mixture of 50% free mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1. First, mRNA was complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, free mRNA was added, and the final concentration was adjusted with Ringer's lactate solution.

Example 2: Vaccination of Cotton Rats with LNP-Formulated mRNA Encoding RSV-F and RSV Cotton Rat Challenge Study

The present Example shows that LNP-formulated mRNA encoding RSV-F induces strong and functional immune responses in cotton rats.

For the development of RSV vaccines the cotton rat is an accepted animal model, especially for the challenge infection. Cotton rats that have received formalin-inactivated RSV virus vaccine preparations respond to RSV infection with enhanced lung pathology. This allows the evaluation of the safety of a vaccination in terms of enhanced disease phenomenon.

To optimize the RSV-specific immune response, mRNA vaccines encoding the pre-fusion stabilized RSV F protein F-del_DSCav were prepared according to Example 1, formulated either with LNPs (see Example 1.4.) or with protamine (see Example 1.5.) and were applied on days 0 and 28 intramuscularly (i.m.) or intradermally (i.d) with different doses of RNA as shown in Table 10. Control animals received a single vaccination at day 0 with 105 pfu live RSV/A2 virus intranasally or two vaccinations at days 0 and 28 with formalin-inactivated RSV virus (FI-RSV) intramuscularly. Additional control animals received buffer only. For the present example, a UTR combination HSD17B4/ALB7 was used, herein referred to as “i-4”.

TABLE 10 Animal groups and vaccination schedule of Example 2 Group Cotton rats Treatment mRNA dose Route Volume 1 5 mRNA/LNP R4745 10 μg i.m. 1 × 100 uL 2 5 mRNA/LNP R4745 2 μg i.m. 1 × 100 uL 3 5 mRNA/LNP R4745 10 μg i.d. 2 × 50 uL 4 5 mRNA/LNP R4745 2 μg i.d. 2 × 50 uL 5 5 mRNA/protamine R5717 80 μg i.m. 1 × 100 uL 6 5 mRNA/protamine R5717 80 μg i.d. 2 × 50 uL 7 5 Live RSV/A2 virus 105 pfu i.n. 1 × 100 uL 8 5 FI-RSV 1:100 i.m. 1 × 100 uL 9 5 Buffer — i.m 1 × 100 uL 10 5 Untreated/uninfected — —

Determination of Virus Neutralization Titers:

Serum was collected at day 49 and RSV virus neutralization titers (VNTs) were measured using a plaque reduction neutralization test (PRNT). Diluted serum samples were incubated with RSV/A2 for 1 hour at room temperature and inoculated in duplicates onto confluent HEp-2 monolayers in 24 well plates. After one hour incubation at 37° C. in a 5% CO2 incubator, the wells were overlayed with 0.75% Methylcellulose medium. After 4 days of incubation, the overlays were removed and the cells were fixed and stained. The corresponding reciprocal neutralizing antibody titers were determined at the 60% reduction end-point of the virus control.

Cotton Rat Challenge Study:

The vaccinated animals were challenged intranasally at day 63 with 105 pfu live RSV/A2 virus in 100 uL. One control group remained untreated and uninfected (group 10). All animals were sacrificed at day 68 and nasal tissue and lung were harvested.

RSV Titers in the Lungs of Challenged Cotton Rats:

Lungs of animals were collected at day 68 (i.e. 5 days after intranasal challenge with 105 pfu live RSV/A2 virus) and RSV/A2 titers were quantified in one lobe by plaque assay. Lung homogenates were clarified by centrifugation and diluted in EMEM. Confluent HEp-2 monolayers were infected in duplicates with diluted homogenates in 24 well plates. After one hour incubation at 37° C. in a 5% CO incubator, the wells were overlayed with 0.75% methylcellulose medium. After 4 days of incubation, the overlays were removed and the cells were fixed and stained with 0.1% crystal violet for one hour and then rinsed and air dried. Plaques were counted and virus titers were expressed as plaque forming units per gram of tissue. Viral titers were calculated as geometric mean±standard error for all animals in a group at a given time.

Lung Histopathology of Challenged Cotton Rats:

Lungs of animals were collected at day 68 (i.e. 5 days after intranasal challenge with 105 pfu live RSV/A2 virus) and one lobe was analyzed by histopathology. Lungs were dissected and inflated with 10% neutral buffered formalin to their normal volume, and then immersed in the same fixative solution. Following fixation, the lungs were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Four parameters of pulmonary inflammation were evaluated: peribronchiolitis (inflammatory cell infiltration around the bronchioles), perivasculitis (inflammatory cell infiltration around the small blood vessels), interstitial pneumonia (inflammatory cell infiltration and thickening of alveolar walls), and alveolitis (cells within the alveolar spaces). Slides were scored blindly on a 0-4 severity scale. The scores were subsequently converted to a 0-100% histopathology scale.

Results:

As can be seen from FIG. 1, the LNP formulated RSV-F (F-del_DSCav1) mRNA vaccines induce the formation of RSV specific functional antibodies in cotton rats as shown by high virus neutralizing antibody titers (groups 1-4). Vaccination with protamine-formulated mRNA induces only weak responses.

As can be seen from FIG. 2, the LNP-formulated RSV-F (F-del_DSCav1) mRNA vaccines reduce lung viral titers in cotton rats challenged with RSV virus and therefore limit RSV infection of the lung (groups 1-4). All the animal groups vaccinated with mRNA formulated in LNPs vaccines showed virus titers below the level of detection of the performed virus titration demonstrating protection of vaccinated cotton rats in terms of viral lung titers. By contrast, the protamine-formulated mRNA vaccines (administrated i.m.) and the Formalin-inactivated virus vaccine reduced only minimally the lung virus titer compared to the buffer control group.

As can be seen from FIG. 3, the lung histopathology analysis from the RSV cotton rat challenge study reveals different pathology scores for the various animal groups. From the histopathology it can be concluded that none of the mRNA vaccinated groups displayed enhanced lung pathology as it is the case for the group that was vaccinated using the formalin-inactivated RSV vaccine. The average pathology scores for peribronchiolitis, perivasculitis, insterstitial pneumonia and alveolitis are much lower for all groups vaccinated with mRNA (groups 1-6) compared to the group with formalin-inactivated RSV (group 8).

Example 3: Vaccination of Cotton Rats with LNP-Formulated mRNA Encoding RSV-F and RSV Cotton Rat Challenge Study

The present Example shows that LNP-formulated mRNA encoding different RSV-F antigens induce strong and functional immune responses in cotton rats for mRNA constructs having UTR combination RPL32/ALB7 (i-2).

This experiment compared mRNA-LNPs encoding three different RSV-F variants: full-length RSV F0, truncated RSV F-del, and pre-fusion stabilized truncated F-del_DSCav1. The mRNA-LNP vaccines were prepared according to Example 1. Cotton rats received two intramuscular vaccinations with mRNA-LNP vaccines on days 0 and 28. Each dose comprised 10 μg or 100 μg mRNA-LNPs. Control animals received a single vaccination at day 0 with 105 pfu live RSV/A2 virus intranasally or two vaccinations at days 0 and 28 with formalin-inactivated RSV virus (FI RSV) intramuscularly. Additional control animals received buffer only (see Table 11).

TABLE 11 Animal groups and vaccination schedule of Example 3 RNA Group Test item ID Dose Route 1 mRNA encoding F0 R3737 10 μg i.m. LNP-formulated 2 mRNA encoding F0 R3737 100 μg i.m. LNP-formulated 3 mRNA encoding F-del R3738 10 μg i.m. LNP-formulated 4 mRNA encoding F-del R3738 100 μg i.m. LNP-formulated 5 mRNA encoding F-delDSCav1 R6122 10 μg i.m. LNP-formulated 6 mRNA encoding F-delDSCav1 R6122 100 μg i.m. LNP-formulated 7 Live RSV/A2 virus 105 pfu i.n. 8 FI RSV 1:100 i.m. 9 Buffer — i.m. 10 untreated/uninfected — —

Determination of Anti-RSV F Protein Antibodies by ELISA:

Blood samples were collected on days 28, 49, and 63 for the determination of anti-RSV F antibody titers. ELISA plates are coated with recombinant human RSV fusion glycoprotein (Extracellular domain of human RSV Fusion glycoprotein (529) fused with a polyhistidine tag at the C-terminus, expressed in Baculovirus-Insect Cells, provided as lyophilized powder). Coated plates are incubated using given serum dilutions. Binding of specific antibodies to the F protein is detected using biotinylated isotype specific anti-mouse antibodies in combination with streptavidin-HRP (horse radish peroxidase) with ABTS substrate.

Determination of Virus Neutralization Titers:

Serum was collected at day 49 and RSV virus neutralization titers (VNTs) were measured using a plaque reduction neutralization test (PRNT). Diluted serum samples were incubated with RSV/A2 for 1 hour at room temperature and inoculated in duplicates onto confluent HEp-2 monolayers in 24 well plates. After one hour incubation at 37° C. in a 5% CO2 incubator, the wells were overlayed with 0.75% Methylcellulose medium. After 4 days of incubation, the overlays were removed and the cells were fixed and stained. The corresponding reciprocal neutralizing antibody titers were determined at the 60% reduction end-point of the virus control.

Cotton Rat Challenge Study:

The vaccinated animals were challenged intranasally at day 63 with 10⁵ pfu live RSV/A2 virus in 100 uL. One control group remained untreated and uninfected. All animals were sacrificed at day 68 and nasal tissue and lung were harvested.

RSV Titers in the Lungs of Challenged Cotton Rats:

Lungs of animals were collected at day 68 (i.e. 5 days after intranasal challenge with 10⁵ pfu live RSV/A2 virus) and RSV/A2 titers were quantified in one lobe by plaque assay as described above.

RSV Titers in Nasal Tissue of Challenged Cotton Rats

Nasal tissue of animals was collected at day 68 (i.e. 5 days after intranasal challenge with 105 pfu live RSV/A2 virus). The nose homogenates were clarified as the lung homogenates by centrifugation and diluted in EMEM. Confluent HEp-2 monolayers were infected in duplicates with diluted homogenates in 24 well plates. After one hour incubation at 37° C. in a 5% CO incubator, the wells were overlayed with 0.75% methylcellulose medium. After 4 days of incubation, the overlays were removed and the cells were fixed and stained with 0.1% crystal violet for one hour and then rinsed and air dried. Plaques were counted and virus titers were expressed as plaque forming units per gram of tissue. Viral titers were calculated as geometric mean±standard error for all animals in a group at a given time.

Lung Histopathology of Challenged Cotton Rats:

Lungs of animals were collected at day 68 (i.e. 5 days after intranasal challenge with 105 pfu live RSV/A2 virus) and one lobe was analyzed by histopathology as described before.

Results:

As can be seen from FIG. 4, all LNP-formulated RSV-F (F0, F-del, F-del_DSCav1) mRNA vaccines induced the formation of RSV specific functional antibodies in cotton rats as shown by high virus neutralizing antibody titers.

FIGS. 5a, 5b and 5c show that LNP-formulated mRNA encoding the RSV F-protein (F0, F-del, and F-del_DSCav1) induces humoral immune responses in cotton rats against the RSV-F protein. Antibody total IgG titers were determined by ELISA. FIG. 5a : Antibody titers determined on day 49; FIGS. 5b and 5c : time-dependent igG titers in serum with 10 μg mRNA (FIG. 5b ) or 100 μg mRNA (FIG. 5c ) encoding the RSV F-protein (F0, F-del, and F-del_DSCav1). With both doses, 10 μg and 100 μg, F0 and F-del vaccines induce higher titers of RSV-F specific IgGs already after prime vaccination (day 28) compared with the vaccination with the live virus. After boost vaccination all tested LNP-formulated RSV-F (F0, F-del, F-del_DSCav1) mRNA vaccines induces humoral immune responses measured with ELISA which are more prominent then the answers of the control vaccinations.

As can be seen from FIG. 6, LNP-formulated RSV-F (F0, F-del, F-del_DSCav1) mRNA vaccines reduced lung viral titers in cotton rats challenged with RSV virus and therefore limit RSV infection of the lung. All the animal groups vaccinated with mRNA formulated in LNPs vaccines showed virus titers below the level of detection of the performed virus titration. The results demonstrate protection of vaccinated cotton rats in terms of viral lung titers. By contrast, the formalin-inactivated virus vaccine (FI RSV) reduced only minimally the lung virus titer compared to the buffer control group.

As can be seen from FIG. 7, the LNP-formulated RSV-F (F0, F-del, F-del_DSCav1) mRNA vaccines strongly reduced viral titers in nasal tissue of cotton rats challenged with RSV virus and therefore reduce RSV infection of the nose. In comparison to the mRNA vaccines the vaccine based on formalin-inactivated virus (F RSV) were not able to reduce the nasal virus titers.

As can be seen from FIG. 8, the lung histopathology analysis from the RSV cotton rat challenge study reveals different pathology scores for the various animal groups. From the histopathology it can be concluded that none of the mRNA vaccinated groups displayed enhanced lung pathology as it is the case for the group that was vaccinated using the formalin-inactivated RSV vaccine. The average pathology scores for peribronchiolitis, perivasculitis, insterstitial pneumonia and alveolitis are much lower for all groups vaccinated with mRNA compared to the group with formalin-inactivated RSV.

To further improve the efficiency of the mRNA-based vaccine, several alternative RSV-F mRNA constructs were designed harboring different UTR combinations to potentially increase translation efficiency of the mRNA. Those mRNA constructs were tested as can be seen in the following Example.

Example 4: In Vitro Expression Screen of RSV-F mRNA Constructs

The present Example shows that the UTR combinations according to the invention strongly improve the expression performance of said mRNA constructs compared to a reference mRNA construct (harboring RPL32/ALB7 UTRs) used e.g. in Example 3 (UTR combination RPL32/ALB7 (i-2)).

To further improve the expression performance of RSV-F mRNA, a screening experiment was conducted to identify advantageous UTR combinations according to the invention. To determine the protein expression performance of RSV-F mRNA constructs comprising different UTR combinations, HEK 293T cells were transfected with different Lipofectamine-formulated mRNA constructs, which all contain the same coding sequence encoding pre-fusion stabilized truncated F-del_DSCav1 but use different 5′- and 3′-UTRs (see Table 12). RSV-F expression was analyzed 24 h after transfection by flow cytometry.

TABLE 12 Overview of mRNA constructs with different UTR combinations used in Example 4 SEQ ID NO: SEQ ID NO: Antigen UTR Design RNA Protein F-del_DSCav1 HSD17B4/PSMB3; a-1 1276 1267 F-del_DSCav1 Ndufa4/PSMB3; a-2 1284 1267 F-del_DSCav1 Slc7a3/PSMB3; a-3 1292 1267 F-del_DSCav1 Nosip/PSMB3; a-4 1300 1267 F-del_DSCav1 Mp68/PSMB3; a-5 1308 1267 F-del_DSCav1 Ubqln2/RPS9; b-1 1316 1267 F-del_DSCav1 ASAH1/RPS9; b-2 1324 1267 F-del_DSCav1 HSD17B4/RPS9; b-3 1332 1267 F-del_DSCav1 HSD17B4/CASP1; b-4 1340 1267 F-del_DSCav1 Nosip/COX6B1; b-5 1348 1267 F-del_DSCav1 Ndufa4/RPS9; c-1 1356 1267 F-del_DSCav1 Nosip/Ndufa1; c-2 1364 1267 F-del_DSCav1 Ndufa4/COX6B1; c-3 1372 1267 F-del_DSCav1 Ndufa4/Ndufa1; c-4 1380 1267 F-del_DSCav1 ATP5A1/PSMB3; c-5 1388 1267 F-del_DSCav1 Rpl31/PSMB3; d-1 1396 1267 F-del_DSCav1 ATP5A1/CASP1; d-2 1404 1267 F-del_DSCav1 Slc7a3/Gnas; d-3 1412 1267 F-del_DSCav1 HSD17B4/Ndufa1; d-4 1420 1267 F-del_DSCav1 Slc7a3/Ndufa1; d-5 1428 1267 F-del_DSCav1 TUBB4B/RPS9; e-1 1436 1267 F-del_DSCav1 Rpl31/RPS9; e-2 1444 1267 F-del_DSCav1 Mp68/RPS9; e-3 1452 1267 F-del_DSCav1 Nosip/RPS9; e-4 1460 1267 F-del_DSCav1 ATP5A1/RPS9; e-5 1468 1267 F-del_DSCav1 ATP5A1/COX6B1; e-6 1476 1267 F-del_DSCav1 ATP5A1/Gnas; f-1 1484 1267 F-del_DSCav1 ATP5A1/Ndufa1; f-2 1492 1267 F-del_DSCav1 HSD17B4/COX6B1; f-3 1500 1267 F-del_DSCav1 HSD17B4/Gnas; f-4 1508 1267 F-del_DSCav1 Mp68/COX6B1; f-5 1516 1267 F-del_DSCav1 Mp68/Ndufa1; g-1 1524 1267 F-del_DSCav1 Ndufa4/CASP1; g-2 1532 1267 F-del_DSCav1 Ndufa4/Gnas; g-3 1540 1267 F-del_DSCav1 Nosip/CASP1; g-4 1548 1267 F-del_DSCav1 Rpl31/CASP1; g-5 1556 1267 F-del_DSCav1 Rpl31/COX6B1; h-1 1564 1267 F-del_DSCav1 Rpl31/Gnas; h-2 1572 1267 F-del_DSCav1 Rpl31/Ndufa1; h-3 1580 1267 F-del_DSCav1 Slc7a3/CASP1; h-4 1588 1267 F-del_DSCav1 Slc7a3/COX6B1; h-5 1596 1267 F-del_DSCav1 Slc7a3/RPS9; i-1 1604 1267 F-del_DSCav1 RPL32(32L4)/ALB7; i-2 1612 1267

Detailed Description of the Transfection and Flow Cytometry Analysis:

293T cells were seeded at a density of 200000 cells/well (200000 cells/2 ml) in a 6-well plate. Each RNA was complexed with Lipofectamine2000 at a ratio of 1/1.5 (w/v) for 20 minutes in Opti-MEM. Lipocomplexed mRNAs were then added to cells for transfection with 2 μg of RNA per well in a total volume of 500 uL. 4 h post start of transfection the transfection solution was exchanged for 2000 uL/well of complete medium. Cells were further maintained at 37° C., 5% CO2 before performing FACS analysis.

24 hours after transfection, expression of antigen of interest was quantified by FACS analysis using standard procedures. Briefly, cells were detached (40 mM Tris HCl pH 7.5 150 mM NaCl, 1 mM EDTA in H20; 5 min at RT), washed with PBS, and stained on the surface with a mouse antibody against the RSV-F (Millipore, Cat: MAB8262) and a fluorescently labeled goat anti-mouse IgG antibody (Sigma, Cat: F5262). Cells were resuspended in 100 uL PFEA buffer (PBS+2% FCS+2 mM EDTA+0.01% NaN3) and analyzed using a BD FACS Canto II. Live/Dead staining was performed with Aqua fluorescent reactive dye (Invitrogen).

The results were compared to the expression from a reference construct of Example 3 containing the RPL32/ALB7 UTR-combination (SEQ ID NO: 1612) which was set to a level of 100%. The results of the analysis are shown in FIG. 9.

Results

As can be seen from FIG. 9, the expression performances of the mRNA constructs comprising UTR combinations according to the invention were strongly increased compared to the construct comprising the reference UTR combination (RPL32/ALB7; i-2).

Example 5: Vaccination of Cotton Rats with LNP-Formulated mRNA Encoding RSV-F and Cotton Rat Challenge Study

Based on the results of the UTR screen of Example 4, mRNA constructs having UTR combinations for optimizing mRNA expression are used in vaccination experiments.

In this experiment mRNA vaccines encoding RSV-F (F, F-del, or F-del_DSCav1) with different UTR combinations according to the invention are compared. The mRNA-LNP vaccines are prepared according to Example 1. Cotton rats receive two intramuscular vaccinations with mRNA-LNP vaccines on days 0 and 28. Each dose comprises 2 μg or 10 μg mRNA-LNPs. Control animals receive a single vaccination at day 0 with 105 pfu live RSV/A2 virus intranasally or formalin-inactivated RSV virus intramuscularly. Additional control animals receive buffer only (see Table 13).

TABLE 13 Animal groups and vaccination schedule of Example 5 SEQ ID NO: Group Test Item 5′-UTR/3′-UTR; RNA Dose Route 1 F0, F-del or F-del_DSCav1 Nosip/RPS9; e-4 286, 701 or 1460 10 μg i.m. 2 F0, F-del or F-del_DSCav1 Nosip/RPS9; e-4 286, 701 or 1460 2 μg i.m. 3 F0, F-del or F-del_DSCav1 Ndufa4/CASP1; g-2 367, 782 or 1532 10 μg i.m. 4 F0, F-del or F-del_DSCav1 Ndufa4/CASP1; g-2 367, 782 or 1532 2 μg i.m. 5 F0, F-del or F-del_DSCav1 Ndufa4/RPS9; c-1 169, 584 or 1356 10 μg i.m. 6 F0, F-del or F-del_DSCav1 Ndufa4/RPS9; c-1 169, 584 or 1356 2 μg i.m. 7 F0, F-del or F-del_DSCav1 Ndufa4/PSMB3; a-2 88, 503 or 1284 10 μg i.m. 8 F0, F-del or F-del_DSCav1 Ndufa4/PSMB3; a-2 88, 503 or 1284 2 μg i.m. 9 F0, F-del or F-del_DSCav1 HSD17B4/PSMB3; a-1 79, 494 or 1276 10 μg i.m. 10 F0, F-del or F-del_DSCav1 HSD17B4/PSMB3; a-1 79, 494 or 1276 2 μg i.m. 11 F0, F-del or F-del_DSCav1 —/muag; i-3 475, 890 or 1628 10 μg i.m. (R6939, R6940 or R5453) 12 F0, F-del or F-del_DSCav1 —/muag; i-3 475, 890 or 1628 2 μg i.m. (R6939, R6940 or R5453) 13 Live RSV/A2 virus 5.0Log IM i.n. 14 UV-Inact. RSV/A2 5.0Log IM i.m. 15 FI-RSV 1:100 i.m. 16 Buffer i.m. 17 untreated/uninfected

Determination of Anti-RSV Immune Responses Using ELISA or PRNT

The induction of anti-RSV immune responses are determined as described before.

Cotton Rat Challenge Study

The vaccinated animals are challenged intranasally at day 63 with 10exp5 pfu live RSV/A2 virus in 100 uL. One control group remains untreated and uninfected. All animals are sacrificed at day 68 and nasal tissue and lung tissue are harvested.

RSV Titers in the Lungs of Challenged Cotton Rats

Lungs of animals are collected at day 68 (i.e. 5 days after intranasal challenge with 10exp5 pfu live RSV/A2 virus) and RSV/A2 titers are quantified in one lobe by plaque assay as described above. In addition, RSV viral genome copy numbers (by measuring copy numbers of the RSV NS-1 gene) and cytokine mRNA levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

RSV Titers in Nasal Tissue of Challenged Cotton Rats

Nasal tissue of animals are collected at day 68 (i.e. 5 days after intranasal challenge with 105 pfu live RSV/A2 virus). The viral titers are quantified as described above. In addition, RSV viral genome copy numbers (by measuring copy numbers of the RSV NS-1 gene) and cytokine mRNA levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Lung Histopathology of Challenged Cotton Rats

Lungs of animals are collected at day 68 (i.e. 5 days after intranasal challenge with 105 pfu live RSV/A2 virus) and one lobe is analyzed by histopathology as described before.

Example 6: Vaccination of Cotton Rats with LNP-Formulated mRNA Encoding RSV-F and Cotton Rat Challenge Study

In this experiment mRNA vaccines encoding RSV-F (F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3 (and/or additionally with F_DSCav1_mut0, _mut4, _mut5, _mut6, _mut7, _mut8 and/or F-del_DSCav1_mut0, _mut4, _mut5, _mut6, _mut7, _mut8) are compared. The mRNA-LNP vaccines are prepared according to Example 1. Cotton rats receive two intramuscular vaccinations with mRNA-LNP vaccines on days 0 and 28. Each dose comprises 2 μg or 10 μg mRNA-LNPs. Control animals receive a single vaccination at day 0 with 105 pfu live RSV/A2 virus intranasally or formalin-inactivated RSV virus intramuscularly. Additional control animals receive buffer only (see Table 14).

TABLE 14 Animal groups of Example 6 RNA SEQ ID NO: SEQ ID NO: Group Test Item ID RNA Protein Route 1 F_DSCav1_mut1 R6771 1997 1636 i.m. 2 F-del_DSCav1_mut1 R6774 2366 2005 i.m. 3 F_DSCav1_mut2 R6772 2735 2374 i.m. 4 F-del_DSCav1_mut2 R6773 3104 2743 i.m. 5 F_DSCav1_mut3 R6770 3473 3112 i.m. 6 F-del_DSCav1_mut3 R6775 3842 3481 i.m. 7 Live RSV/A2 virus i.n. 8 UV-Inact. RSV/A2 i.m. 9 FI-RSV i.m. 10 Buffer i.m. 11 Untreated/uninfected

Determination of Anti-RSV Immune Responses Using ELISA or PRNT

The induction of anti-RSV immune responses are determined as described before.

Cotton Rat Challenge Study

The vaccinated animals are challenged intranasally at day 63 with 10exp5 pfu live RSV/A2 virus in 100 uL. One control group remains untreated and uninfected. All animals are sacrificed at day 68 and nasal tissue and lung tissue are harvested.

RSV Titers in the Lungs of Challenged Cotton Rats

Lungs of animals are collected at day 68 (i.e. 5 days after intranasal challenge with 10exp5 pfu live RSV/A2 virus) and RSV/A2 titers are quantified in one lobe by plaque assay as described above. In addition, RSV viral genome copy numbers (by measuring copy numbers of the RSV NS-1 gene) and cytokine mRNA levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

RSV Titers in Nasal Tissue of Challenged Cotton Rats

Nasal tissue of animals are collected at day 68 (i.e. 5 days after intranasal challenge with 105 pfu live RSV/A2 virus). The viral titers are quantified as described above. In addition, RSV viral genome copy numbers (by measuring copy numbers of the RSV NS-1 gene) and cytokine mRNA levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Lung Histopathology of Challenged Cotton Rats Lungs of animals are collected at day 68 (i.e. 5 days after intranasal challenge with 10⁵ pfu live RSV/A2 virus) and one lobe is analyzed by histopathology as described before.

Example 7: Vaccination of Cotton Rats with SNP-Formulated mRNA Encoding RSV-F and a Further RSV Antigen and Cotton Rat Challenge Study

To broaden and optimize the RSV-specific immune response, especially to increase T-cell dependent immune responses, mRNA vaccines encoding different RSV proteins (RSV F (F0, F, F-del, F_DSCav1 F-del_DSCav1, or F_DSCav1 mut0-mut8 or F-del_DSCav1 mut0-mut8) are prepared according to Example 1. In order to assess the effect of single or combined vaccines, these vaccines are administered either alone or in combination as shown in Table 9. Cotton rats receive two intramuscular (i.m.) vaccinations with mRNA-LNP vaccines on days 0 and 28 with 2 μg or 10 μg mRNA for each antigen. Additional groups are immunized intramuscularly (i.m.) with formalin-inactivated RSV and/or live RSV/A2 to compare their immunogenicity to mRNA vaccines. Additional control animals receive buffer only (see Table 15). The RSV-specific immune responses are determined by Elisa or Intracellular cytokine staining (ICS).

TABLE 15 Animal groups of Example 7 SEQ ID NO: Group Test Item RNA Route 1 F 475, 890, 1259, 1628, 1997, 2366, 2735, 3104, 3473, 3842, 4211, i.m. 4580, 4949, 5318, 5687, 6056, 6425, 6794, 7163, 7532, 7901, or 8270 2 M 10046 i.m. 3 N 10496 i.m. 4 M2-1 11545 i.m. 5 P 10999 i.m. 6 F + M F (SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366, 2735, 3104, i.m. 3473, 3842, 4211, 4580, 4949, 5318, 5687, 6056, 6425, 6794, 7163, 7532, 7901, or 8270) + M (SEQ ID NO: 10046) 7 F + N F (SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366, 2735, 3104, i.m. 3473, 3842, 4211, 4580, 4949, 5318, 5687, 6056, 6425, 6794, 7163, 7532, 7901, or 8270) + N (SEQ ID NO: 10496) 8 F + M2-1 F (SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366, 2735, 3104, i.m. 3473, 3842, 4211,4580, 4949, 5318, 5687, 6056, 6425, 6794, 7163, 7532, 7901, or 8270) + M2-1 (SEQ ID NO: 11545) 9 F + P F (SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366, 2735, 3104, i.m. 3473, 3842, 4211, 4580, 4949, 5318, 5687, 6056, 6425, 6794, 7163, 7532, 7901, or 8270) + P (SEQ ID NO: 10999) 10 F + M + P F (SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366, 2735, 3104, i.m. 3473, 3842, 4211, 4580, 4949, 5318, 5687, 6056, 6425, 6794, 7163, 7532, 7901, or 8270) + P (SEQ ID NO: 10999) + M (SEQ ID NO: 10046). 11 F + M + N + P F (SEQ ID NOs: 475, 890, 1259, 1628, 1997, 2366, 2735, 3104, i.m. 3473, 3842, 4211, 4580, 4949, 5318, 5687, 6056, 6425, 6794, 7163, 7532, 7901, or 8270) + P (SEQ ID NO: 10999) + M (SEQ ID NO: 10046) + N (SEQ ID NO: 10496) 12 Live RSV/A2 virus i.n. 13 UV-Inact. RSV/A2 i.m. 14 FI-RSV i.m. 15 Buffer i.m. 16 Untreated/uninfected

For vaccination experiments of Example 7, polyvalent mRNA compositions/vaccines are produced (e.g. groups 6-10) according to procedures as disclosed in the PCT application WO2017/109134 using at least two different sequence optimized DNA templates (each of which generated as described in Example 1). In short, a DNA construct mixture (each of which comprising a different coding sequences and a T7 promotor) is used as a template for simultaneous RNA in vitro transcription to generate a mixture of mRNA constructs. Subsequently, the obtained RNA mixture is used for co-purification using RP-HPLC. Following that, the obtained purified RNA mixture is formulated in LNPs (as described in section 1.4.) to generate a polyvalent LNP-formulated RNA composition/vaccine.

Determination of Anti-RSV Immune Responses Using ELISA PRNT:

The induction of anti-RSV immune responses are determined as described before, with suitable recombinant proteins or peptides for its detection.

Intracellular Cytokine Staining (ICS):

Splenocytes from vaccinated and control mice are isolated according to a standard protocol. Briefly, isolated spleens are grinded through a cell strainer and washed in PBS/1% FBS followed by red blood cell lysis. After an extensive washing step with PBS/1% FBS splenocytes are seeded into 96-well plates (2×106 cells/well). The next day cells are stimulated with a suitable RSV peptide or an irrelevant control peptide and 2.5 μg/ml of an anti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in the presence of the mixture of GolgiPlug™/GolgiStop™ (Protein transport inhibitors containing Brefeldin A and Monensin, respectively; BD Biosciences). After stimulation cells are washed and stained for intracellular cytokines using the Cytofix/Cytoperm reagent (BD Biosciences) according to the manufacturer's instructions. The following antibodies are used for staining: CD8-PECy7 (1:200), CD3-FITC (1:200), IL2-PerCP-Cy5.5 (1:100), TNFα-PE (1:100), IFNγ-APC (1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) and incubated with Fcy-block diluted 1:100. Aqua Dye is used to distinguish live/dead cells (Invitrogen). Cells are collected using a Canto I flow cytometer (Beckton Dickinson). Flow cytometry data are analysed using FlowJo software (Tree Star, Inc.).

Cotton Rat Challenge Study:

The vaccinated animals are challenged intranasally at day 63 with 10exp5 pfu live RSV/A2 virus in 100 uL. One control group remains untreated and uninfected. All animals are sacrificed at day 68 and nasal tissue and lung tissue are harvested.

Cotton Rat Challenge Study:

The vaccinated animals are challenged intranasally at day 63 with 10exp5 pfu live RSV/A2 virus in 100 uL. One control group remains untreated and uninfected. All animals are sacrificed at day 68 and nasal tissue and lung tissue are harvested. Serum is collected at day 49 and RSV virus neutralization titers (VNTs) are measured using a plaque reduction neutralization test (PRNT) as described in Example 2.

RSV Titers in the Lungs of Challenged Cotton Rats:

Lungs of animals are collected at day 68 (i.e. 5 days after intranasal challenge with 10exp5 pfu live RSV/A2 virus) and RSV/A2 titers are quantified in one lobe by plaque assay as described above. In addition, RSV viral genome copy numbers (by measuring copy numbers of the RSV NS-1 gene) and cytokine mRNA levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

RSV Titers in Nasal Tissue of Challenged Cotton Rats:

Nasal tissue of animals are collected at day 68 (i.e. 5 days after intranasal challenge with 105 pfu live RSV/A2 virus). The viral titers are quantified as described above. In addition, RSV viral genome copy numbers (by measuring copy numbers of the RSV NS-1 gene) and cytokine mRNA levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Lung Histopathology of Challenged Cotton Rats:

Lungs of animals are collected at day 68 (i.e. 5 days after intranasal challenge with 105 pfu live RSV/A2 virus) and one lobe is analyzed by histopathology as described before.

Example 8: Expression of Different RSV F Proteins in HeLa Cells and Analysis by FACS

To determine in vitro protein expression of the mRNA constructs, HeLa cells were transiently transfected with mRNA encoding RSV F antigens and stained for RSV Fusing a suitable anti-F protein antibodies (raised in mouse), counterstained with a FITC-coupled secondary anti-mouse antibody (F5262 from Sigma). HeLa cells were seeded in a 6-well plate at a density of 400,000 cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1% Pen/Strep), 24 h prior to transfection. HeLa cells were transfected with 2 μg unformulated mRNA using Lipofectamine 2000 (Invitrogen). The mRNA constructs prepared according to Example 1 and listed in Table 16 were used in the experiment (see also Table 9), including a negative control (water for injection). 24 hours post transfection, HeLa cells were stained with suitable anti RSV-F antibodies (raised in mouse; 1:500) and anti-mouse FITC labelled secondary antibody (1:500) and subsequently analyzed by flow cytometry (FACS) on a BD FACS Canto II using the FACS Diva software. Quantitative analysis of the fluorescent FITC signal was performed using the FlowJo software package (Tree Star, Inc.). Cells were stained intracellularly or alternatively on the cell surface. The results are shown in FIGS. 10a and 10b , representing two different independent experiments.

TABLE 16 Overview of mRNA constructs used in Example 8 RNA SEQ ID NO: SEQ ID NO: see ID Antigen UTR Design RNA Protein Figure R6939 F0 —/muag; i-3 475 68 10a, 10b R6808 F0_DSCav1 —/muag; i-3 1259 898 10a, 10b R6770 F_DSCav1_mut3 —/muag; i-3 3473 3112 10a R6771 F_DSCav1_mut1 —/muag; i-3 1997 1636 10a R6772 F_DSCav1_mut2 —/muag; i-3 2735 2374 10a R6940 F-del —/muag; i-3 890 483 10a, 10b R5453 F-del_DSCav1 —/muag; i-3 1628 1267 10a, 10b R6775 F-del_DSCav1_mut3 —/muag; i-3 3842 3481 10a R6774 F-del_DSCav1_mut1 —/muag; i-3 2366 2005 10a R6773 F-del_DSCav1_mut2 —/muag; i-3 3104 2743 10a R7454 F_DSCav1_mut0 —/muag; i-3 4211 3850 10b R7456 F_DSCav1_mut5 —/muag; i-3 5687 5326 10b R7458 F_DSCav1_mut4 —/muag; i-3 4949 4588 10b R7455 F-del_DSCav1_mut0 —/muag; i-3 4580 4219 10b R7457 F-del_DSCav1_mut5 —/muag; i-3 6056 5695 10b R7459 F-del_DSCav1_mut4 —/muag; i-3 5318 4957 10b

Results:

The results show that the used mRNA constructs led to a detectable intracellular RSV F protein expression as well as to a detectable protein expression at the cell surface. The RSV F constructs with deleted C-terminus (F-del) (e.g. R6940, R5453, R6775, R6774, R6773, R7455, R7457, R7459) show an increased cell surface protein expression in comparison to F full length constructs. The construct encoding F-del_DSCav1_mut0 (R7455) exhibits the highest expression.

The results exemplify that the inventive mRNA encoding F proteins (F0, F-del, F0_DSCav1, F-del_DSCav1, F_DSCav1_mut0, F-del_DSCav1_mut0, F_DSCav1_mut1, F-del_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut2, F_DSCav1_mut3, F-del_DSCav1_mut3, F_DSCav1_mut4, F-del_DSCav1_mut4, F_DSCav1_mut5, F-del_DSCav1_mut5) is translated in cells and are present at the cell surface, which is a prerequisite for an mRNA-based RSV vaccine.

Example 9: Vaccination of Cotton Rats with LNP-Formulated mRNA Encoding RSV-F and RSV Cotton Rat Challenge Study

The present Example shows that LNP-formulated mRNA according to the invention encoding different pre-fusion stabilized RSV-F antigens induce strong, functional and protective immune responses in cotton rats.

This experiment compared mRNA-LNPs encoding different pre-fusion stabilized RSV-F variants (mut 1, mut2, mut3) with either full-length F or with the truncated F (F-del). The mRNA-LNP vaccines were prepared according to Example 1. Cotton rats received two intramuscular vaccinations with mRNA-LNP vaccines on days 0 and 28. Each dose comprised 100 μg mRNA-LNPs. Control animals received a single vaccination at day 0 with 105 pfu live RSV/A2 virus or UV inactivated RSV/A2 virus or two vaccinations at days 0 and 28 with formalin-inactivated RSV virus (F1 RSV) intramuscularly. Additional control animals received buffer only (see Table 17).

TABLE 17 Animal groups and vaccination schedule of Example 9 RNA SEQ ID NO: SEQ ID NO: Group Test item ID RNA Protein Dose Route 1 mRNA encoding F0 R6939 475 68 100 μg i.m. LNP-formulated 2 mRNA encoding R6808 1259 898 100 μg i.m. F0_DSCav1 LNP-formulated 3 mRNA encoding R6770 3473 3112 100 μg i.m. F_DSCav1_mut3 LNP-formulated 4 mRNA encoding R6771 1997 1636 100 μg i.m. F_DSCav1_mut1 LNP-formulated 5 mRNA encoding R6772 2735 2374 100 μg i.m. F_DSCav1_mut2 LNP-formulated 6 mRNA encoding F-del R6940 890 483 100 μg i.m. LNP-formulated 7 mRNA encoding R5453 1628 1267 100 μg i.m. F-del_DSCav1 LNP-formulated 8 mRNA encoding R6775 3842 3481 100 μg i.m. F-del_DSCav1_mut3 LNP-formulated 9 mRNA encoding R6774 2366 2005 100 μg i.m. F-del_DSCav1_mut1 LNP-formulated 10 mRNA encoding R6773 3104 2743 100 μg i.m. F-del_DSCav1_mut2 LNP-formulated 11 Live RSV/A2 virus 10⁵ i.m. 12 UV-inact. RSV/A2 10⁵ i.m. 13 FI RSV 1:100 i.m. 14 Buffer i.m. 15 untreated/uninfected i.m.

Determination of Anti-RSV F Protein Antibodies by ELISA:

Blood samples were collected on days 0, 28, 49, and 63 for the determination of anti-RSV F antibody titers. ELISA plates were coated with purified F protein extracted from RSV/A2-infected HEp-2 cells or methanol/Acetone fixed RSV/B infected HEp-2 cells. Coated plates were incubated using given serum dilutions. Binding of specific antibodies to the F protein was detected using isotype specific anti-cotton rat antibodies in combination with streptavidin-HRP (horse radish peroxidase) with TMB substrate.

Determination of Virus Neutralization Titers:

Serum was collected on days 0, 28, 49, and 63 and RSV virus neutralization titers (VNTs) were measured using a plaque reduction neutralization test (PRNT). Diluted serum samples were incubated with RSV/A2 or RSV/B (25-50 PFU) for 1 hour at room temperature and inoculated in duplicates onto confluent HEp-2 monolayers in 24 well plates. After one hour incubation at 37° C. in a 5% CO2 incubator, the wells were overlayed with 0.75% Methylcellulose medium. After 4 days of incubation, the overlays were removed and the cells were fixed and stained. The corresponding reciprocal neutralizing antibody titers were determined at the 60% reduction end point of the virus control.

Cotton Rat Challenge Study:

The vaccinated animals were challenged intranasally at day 63 with 10⁵ pfu live RSV/A2 virus in 100 uL. One control group remained untreated and uninfected. All animals were sacrificed at day 68 and nasal tissue and lung were harvested.

RSV Titers in Nasal Tissue of Challenged Cotton Rats

Lungs and nasal tissue of animals were collected at day 68 (i.e. 5 days after intranasal challenge with 10⁵ pfu live RSV/A2 virus). The nose homogenates were clarified as the lung homogenates by centrifugation and diluted in EMEM. Confluent HEp-2 monolayers were infected in duplicates with diluted homogenates in 24 well plates. After one hour incubation at 37° C. in a 5% CO incubator, the wells were overlayed with 0.75% methylcellulose medium. After 4 days of incubation, the overlays were removed and the cells were fixed and stained with 0.1% crystal violet for one hour and then rinsed and air dried. Plaques were counted and virus titers were expressed as plaque forming units per gram of tissue.

Pulmonary Histopathology

Lungs were dissected and inflated with 10% neutral buffered formalin to their normal volume, and then immersed in the same fixative solution. Following fixation, the lungs were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Four parameters of pulmonary inflammation were evaluated: peribronchiolitis (inflammatory cell infiltration around the bronchioles), perivasculitis (inflammatory cell infiltration around the small blood vessels), interstitial pneumonia (inflammatory cell infiltration and thickening of alveolar walls), and alveolitis (cells within the alveolar spaces). Slides are scored blind on a 0-4 severity scale. The scores are subsequently converted to a 0-100% histopathology scale.

Results:

FIG. 11 shows that all tested LNP-formulated mRNA constructs encoding different RSV F proteins (F0, F0_DSCav1, F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del, F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) according the invention induce humoral immune responses in cotton rats against the RSV-F protein. Antibody total IgG titers were determined by ELISA.

The mRNA constructs encoding the truncated F (F-del) (F-del, F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) induced higher and faster responses than the constructs with the full length F sequence on d28, no significant difference is detectable at later time points. Stabilized constructs (F0_DSCav1, F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) initially yield lower ELISA titers than wildtype constructs (F0 and F-del). Levels are not significantly different at later time points.

With a dose of 100 μg, the constructs encoding the stabilized and truncated F protein (F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) induce higher titers of RSV-F specific IgGs already after prime vaccination (day 28) compared with the vaccination with the live virus. After boost vaccination all tested LNP-formulated RSV-F mRNA vaccines induces humoral immune responses measured with ELISA which are more prominent then the answers of the control vaccinations.

As can be seen from FIG. 12, all LNP-formulated RSV-F (F0, F0_DSCav1, F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del, F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) mRNA vaccines induced the formation of RSV specific functional antibodies in cotton rats as shown by high virus neutralizing antibody titers. The LNP-formulated RSV-F truncated constructs (F-del, F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) induced higher and faster responses than F full length constructs. The constructs for pre-fusion stabilization according to the invention (F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) induce higher responses than F0, F0_DSCav1, F-del, F-del_DSCav1.

As can be seen from FIG. 13, the lung histopathology analysis from the RSV cotton rat challenge study reveals different pathology scores for the various animal groups. From the histopathology it can be concluded that none of the mRNA vaccinated groups displayed enhanced lung pathology as it is the case for the group that was vaccinated using the formalin-inactivated RSV vaccine. The average pathology scores for peribronchiolitis are lower for all groups vaccinated with mRNA compared to the group with formalin-inactivated RSV. The LNP-formulated RSV-F truncated constructs (F-del, F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) showed in general lower pathology scores than F full length constructs. The constructs for pre-fusion stabilization according to the invention (F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) yielded better responses (lower pathology scores) than F0, F0_DSCav1, F-del, F-del_DSCav1.

As can be seen from FIG. 14a , all in this experiment tested LNP-formulated RSV-F mRNA vaccines reduced lung viral titers in cotton rats challenged with RSV virus and therefore limit RSV infection of the lung. All the animal groups vaccinated with mRNA formulated in LNPs vaccines showed virus titers below the level of detection of the performed virus titration. The results demonstrate protection of vaccinated cotton rats in terms of viral lung titers. By contrast, the formalin-inactivated virus vaccine (FI RSV) and the UV-inactivated RSV/A2 virus control reduced only minimally the lung virus titer compared to the buffer control group.

As can be seen from FIG. 14b , the LNP-formulated RSV-F mRNA vaccines strongly reduced viral titers in nasal tissue of cotton rats challenged with RSV virus and therefore reduce RSV infection of the nose. In comparison to the mRNA vaccines the vaccine based on formalin-inactivated virus (FI RSV) and the UV-inactivated RSV/A2 virus control were not able to reduce the nasal virus titers. A complete protection of the nose was only achieved by the pre-fusion stabilization according to the invention (F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2).

To conclude, the results show that LNP-formulated RNA constructs encoding RSV-F truncated proteins (F-del, F-del_DSCav1, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2) are in general more immunogenic than RNA constructs encoding for full length F proteins. Constructs featuring Ds-Cav1 mutations alone (F0_DSCav1 and F-del_DSCav1) are suboptimal as an mRNA based vaccine in comparison with the constructs encoding the further stabilized F proteins according the invention (F_DSCav1_mut3, F_DSCav1_mut1, F_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut1, F-del_DSCav1_mut2).

To further improve the efficiency of the mRNA-based vaccine, several alternative RSV-F mRNA constructs were designed harboring further mutations stabilizing the pre-fusion conformation in combination with the beneficial truncated F-del protein. Those mRNA constructs were tested as can be seen in the following Example.

Example 10: Vaccination of Cotton Rats with LNP-Formulated mRNA Encoding RSV-F and RSV Cotton Rat Challenge Study

The present Example shows that LNP-formulated mRNA according to the invention encoding different pre-fusion stabilized RSV-F antigens induce strong, functional and protective immune responses in cotton rats.

The mRNA-LNP vaccines were prepared according to Example 1. Cotton rats received either one or two intramuscular vaccinations with mRNA-LNP vaccines on day 0 or on days 0 and 28. Each dose comprised 100 μg mRNA-LNPs. Control animals received a single vaccination at day 28 with 10⁵ pfu live RSV/A2 virus or formalin-inactivated RSV virus (FI RSV) intramuscularly. Additional control animals received buffer only (see Table 18).

TABLE 18 Animal groups and vaccination schedule of Example 10 RNA SEQ ID NO: SEQ ID NO: Group Test item ID RNA Protein Dose Immunization Route 1 (1) mRNA encoding F-del R6940 890 483 100 μg day 0, day 28 i.m. LNP-formulated 2 (2) mRNA encoding R6773 3104 2743 100 μg day 0, day 28 i.m. F-del_DSCav1_mut2 LNP-formulated 3 (3) mRNA encoding R7455 4580 4219 100 μg day 0, day 28 i.m. F-del_DSCav1_mut0 LNP-formulated 4 (4) mRNA encoding R7457 6056 5695 100 μg day 0, day 28 i.m. F-del_DSCav1_mut5 LNP-formulated 5 (5) mRNA encoding R7459 5318 4957 100 μg day 0, day 28 i.m. F-del_DSCav1_mut4 LNP-formulated 6 (1) mRNA encoding F-del R6940 890 483 100 μg day 28 i.m. LNP-formulated 7 (2) mRNA encoding R6773 3104 2743 100 μg day 28 i.m. F-del_DSCav1_mut2 LNP-formulated 8 (3) mRNA encoding R7455 4580 4219 100 μg day 28 i.m. F-del_DSCav1_mut0 LNP-formulated 9 (4) mRNA encoding R7457 6056 5695 100 μg day 28 i.m. F-del_DSCav1_mut5 LNP-formulated 10 (5) mRNA encoding R7459 5318 4957 100 μg day 28 i.m. F-del_DSCav1_mut4 LNP-formulated 11 Live RSV/A2 virus 10⁵ day 28 i.m. 12 FI RSV 1:100 day 28 i.m. 13 Buffer day 0, day 28 i.m. 14 untreated/uninfected i.m.

Determination of Anti-RSV F Protein Antibodies by ELISA and Virus Neutralization Titers

Blood samples were collected on days 0, 28, 49, and 63 for the determination of anti-RSV F antibody titers and RSV virus neutralization titers (VNTs) measured using the plaque reduction neutralization test (PRNT) as described before (see Example 9).

Cotton Rat Challenge Study:

The vaccinated animals were challenged intranasally at day 63 with 10⁵ pfu live RSV/A2 virus in 100 uL. One control group remained untreated and uninfected. All animals were sacrificed at day 68 and nasal tissue and lung were harvested. The RSV titers in nasal tissue of challenged cotton rats and the pulmonary histopathology are analyzed as described before (see Example 9).

Results:

FIG. 15 shows that all tested LNP-formulated mRNA constructs encoding different RSV F proteins (F-del, F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) according the invention induce humoral immune responses in cotton rats against the RSV-F protein. Antibody total IgG titers were determined by ELISA. RNA constructs encoding stabilized F proteins (F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) initially yield lower ELISA titers than the construct encoding the truncated wildtype F protein, F-del. This trend is also detectable at later time points by using higher serum dilutions for the ELISA (data not shown). The IgG response can be significantly boosted by a second immunization.

As can be seen from FIG. 16, all LNP-formulated RSV-F (F-del, F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) mRNA vaccines induced the formation of RSV specific functional antibodies in cotton rats as shown by high virus neutralizing antibody titers. The LNP-formulated constructs for pre-fusion stabilization according to the invention (F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) induce higher or comparable responses than F-del. The RNA construct R7457 coding for the pre-fusion stabilized protein F-del_DSCav1_mut5 induces the highest VNTs (comparable to the best results induced by best pre-fusion stabilized construct in the previous experiment (Example 9, R6773).

The lung histopathology analysis shows that none of the mRNA vaccinated groups displayed enhanced lung pathology as it is the case for the group that was vaccinated using the formalin-inactivated RSV vaccine (see FIG. 17a (for Peribronciolitis and Perivasculitis) and FIG. 17b (for Interstitial Pneumonia and Alveolitis)). The constructs for pre-fusion stabilization according to the invention (F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) yielded better responses (lower pathology scores) than F-del. Animals received two vaccinations showed a lower pathology score in comparison to animals only received one vaccination.

As can be seen from FIG. 18a , all in this experiment tested LNP-formulated RSV-F mRNA vaccines reduced lung viral titers in cotton rats challenged with RSV virus and therefore limit RSV infection of the lung. This is true even for groups receiving only one vaccination. The results demonstrate protection of vaccinated cotton rats in terms of viral lung titers. By contrast, the formalin-inactivated virus vaccine (FI RSV) reduced only minimally the lung virus titer compared to the buffer control group.

As can be seen from FIG. 18b , the LNP-formulated RSV-F mRNA vaccines strongly reduced viral titers in nasal tissue of cotton rats challenged with RSV virus and therefore reduce RSV infection of the nose. In comparison to the mRNA vaccines the vaccine based on formalin-inactivated virus (FI RSV) was not able to reduce the nasal virus titers. A complete protection of the nose was only achieved for some groups received two vaccinations (e.g. for R7457 (F-del_DSCav_mut4) and R7459 (F-del_DSCav_mut5)).

To conclude, the results show that LNP-formulated RNA constructs encoding pre-fusion stabilized RSV-F truncated proteins (F-del_DSCav1_mut2, F-del_DSCav1_mut0, F-del_DSCav1_mut5, F-del_DSCav1_mut4) raised in general higher functional titers and achieved a more pronounced protection against infection in comparison with the RNA construct coding for the wildtype truncated F protein F_del. The best results were reached by RNA constructs encoding F-del_DSCav1_mut2 or F-del_DSCav1_mut5 (R6773, R7457).

Example 11: Expression of Different RSV T-Cell Antigens Using a Rabbit Reticulocyte Lysate System

To determine in vitro protein expression of the mRNA constructs according to the second aspect of the invention (mRNA constructs coding for RSV matrix protein M, phosphoprotein P, nucleoprotein N and matrix protein M2-1 protein), unformulated RNAs (prepared according to Example 1) were mixed with components of Promega Rabbit Reticulocyte Lysate System according to manufacture protocol. The lysate contains the cellular components necessary for protein synthesis (tRNA, ribosomes, amino acids, initiation, elongation and termination factors). As positive control Luciferase RNA from Lysate System Kit was used. The translation result was analyzed by SDS-Page and Western Blot analysis (IRDye 800CW streptavidin antibody (1:2000)). Table 19 summarizes the tested RNA constructs.

TABLE 19 Overview of mRNA constructs used in Example 11 RNA SEQ ID NO: SEQ ID NO: Predicted ID Antigen UTR Design RNA Protein kDa R7595 M —/muag; i-3 10046 9684 R7596 P —/muag; i-3 10999 10637 27.1 R7597 N —/muag; i-3 10496 10134 43.4 R7598 M2-1 —/muag; i-3 11545 11183 22.1 Luc 61

Results:

The results (see FIG. 19) show that the used mRNA constructs led to a detectable protein expression, which is a prerequisite for an mRNA-based RSV vaccine (size shifts of RSV P and M2-1 on the gel may be due to phosphorylation as described e.g. in Richard et al., 2018 or in Hardy and Wertz, 2000 PMIDs: 29489893 and 10846068).

Example 12: Vaccination of Mice with LNP-Formulated mRNA Encoding Different RSV T-Cell Antigens Alone and in the Combination with RSV F (F-Del)

The present Example analyses LNP-formulated mRNA encoding different RSV T-cell antigens according to the second aspect of the invention (RSV M, P, N, and M2-1) alone or in combination with LNP formulated mRNA encoding the truncated RSV F (F-del). The mRNA-LNP vaccines were prepared according to Example 1. Balb/c mice received two intramuscular vaccinations with mRNA-LNP vaccines on days 0 and 28. Each dose comprised 5 μg, 2.5 μg or 2.5 μg+2.5 μg mRNA-LNPs (for groups receiving mRNAs encoding different antigens the LNPs were separately formulated and mixed before administration). Control animals received buffer only (see Table 20).). Humoral, as well as cellular immune responses and neutralizing antibodies were analyzed by ELISA, FACS, PRNT and ICS.

TABLE 20 Animal groups and vaccination schedule of Example 12 RNA SEQ ID NO: SEQ ID NO: Group Test item ID RNA Protein Dose Route 1 NaCl buffer — — — i.m. 2 M: mRNA encoding RSV M R7595 10046  9684 5 μg i.m. LNP-formulated 3 P: mRNA encoding RSV P R7596 10999 10637 5 μg i.m. LNP-formulated 4 N: mRNA encoding RSV N R7597 10496 10134 5 μg i.m. LNP-formulated 5 M2-1: mRNA encoding M2-1 R7598 11545 11183 5 μg i.m. LNP-formulated 6 F: mRNA encoding F-del R6940  890  483 2.5 μg i.m. LNP-formulated 7 F + M: mRNA encoding F-del + R7595 + 890 + 483 + 2.5 μg + i.m. mRNA encoding RSV M R6940 10046 9684 2.5 μg LNP-formulated 8 F + P: mRNA encoding F-del + R7596 + 890 + 483 + 2.5 μg + i.m. mRNA encoding RSV P R6940 10999 10637 2.5 μg LNP-formulated 9 F + N: mRNA encoding F-del + R7597 + 890 + 483 + 2.5 μg + i.m. mRNA encoding RSV N R6940 10496 10134 2.5 μg LNP-formulated 10 F + M2-1: mRNA encoding F-del + R7598 + 890 + 483 + 2.5 μg + i.m. mRNA encoding RSV M2-1 R6940 11545 11183 2.5 μg LNP-formulated

Determination of Anti-RSV F Protein Antibodies by ELISA:

Blood samples were collected on days 0, 14, 28, and 49 for the determination of anti-RSV F antibody titers. ELISA plates were coated with recombinant human RSV fusion glycoprotein (hRSV F Protein (A2; His-tag; Sino Biological; Cat: 11049-V08B)). Coated plates were incubated using given serum dilutions. Binding of specific antibodies to the F protein was detected using isotype specific anti-mouse rat antibodies in combination with streptavidin-HRP (horse radish peroxidase) and the Amplex® UltraRed Reagent (Invitrogen (Ref. No. A36006).

Detection of Antigen-Specific Humoral Antibody Responses Measured by FAGS

Hela cells were transfected with 2 μg RSV mRNA constructs (R7595, R7596, R7597, R7598) using lipofectamine. The cells were harvested 20 h post transfection, and seeded at 1×10⁵ per well into a 96 well plate. The cells were incubated with corresponding sera of vaccinated mice (serum of day 28 and 49, diluted 1:50) followed by a FITC-conjugated anti-mouse IgG antibody staining. Cells were acquired on BD FACS Canto II using DIVA software and analyzed by FlowJo. The results are shown in FIG. 21a (day 28) and FIG. 21b (day 49). As read out the percentage of FITC-positive cells (out of live cells) was used.

Determination of Virus Neutralization Titers:

Serum was collected on day 49 and RSV virus neutralization titers (VNTs) were measured using a plaque reduction neutralization test (PRNT). Diluted serum samples were incubated with RSV/A2 (25-50 PFU) for 1 hour at room temperature and inoculated in duplicates onto confluent HEp-2 monolayers in 24 well plates. After one hour incubation at 37° C. in a 5% CO2 incubator, the wells were overlayed with 0.75% Methylcellulose medium. After 4 days of incubation, the overlays were removed and the cells were fixed and stained. The corresponding reciprocal neutralizing antibody titers were determined at the 60% reduction end-point of the virus control.

Determination of Cellular Immune Responses in Mice by ICS Using FACS

Splenocytes and lung cells from vaccinated and control mice were isolated according to standard protocols. Briefly, isolated spleens were grinded through a cell strainer and washed in PBS (1% FCS). After centrifugation, the cell pellet was resuspended with the remaining supernatant, treated with red cell lysis buffer and washed repeatedly with PBS (1% FCS). Finally, splenocytes were resuspended in FCS/10% DMSO, transferred into a cryovial and stored at −80° C. Lungs were perfused using RPMI-1640 medium (10% FCS; 2 mM glutamine; 1% Pen/Strep), harvested, and then stored in RPMI-1640 medium (10% FCS; 2 mM glutamine; 1% Pen/Strep) at 4° C. until lung cells were isolated using the Lung Dissociation Kit mouse from Miltenyi Biotec according to the manufacturer's instructions. For ICS, 2×10⁶ cells (splenocytes or lung cells) were seed in duplicates in round-bottom plates and stimulated with RSV M2-1-specific (VYNTVISYI, SYIGSINNI, SYIESNRKN, KSIDTLSEI), RSV F-specific (FYQSTCSAV, TYMLTNSEL, KYKNAVTEL, KIMTSKTDV), RSF M-specific (KHTATRFAI, AQMPSKFTI, KYIKPQSQF, TYLRSISVR), RSV P-specific (SPITSNSTI, SYEEINDQT), or RSV N-specific (SYKKMLKEM, FYHILNNPK, KYTIQRSTG, GYHVKANGV) peptides. After Golgi block (GolgiPlug 1/1000) and incubation for intracellular cytokine accumulation, cells were subjected to live/dead staining using Aqua-dye. Then, cells were surface stained using the antibodies α-CD8-APC-H7 (1/100), α-CD4-BD-Horizon V450 (1/200), α-Thy1.2-FITC (1/300), and α-CD107a PE-Cy7 (1/100) for splenocytes and α-CD8-APC-H7 (1/100), α-CD4-BD-Horizon V450 (1/200), α-Thy1.2-FITC (1/300), α-CD69-PE (1/100), and α-CD103-PE-Cy7 (1/60) for lung cells. After treatment with Cytofix/Cytoperm, lung cells were intracellularly stained with α-IFNg-APC (1/100) and splenocytes additionally with α-TNFα-PE (1/100). Finally, cells were acquired on BD FACS Canto II using DIVA software and analyzed by FlowJo.

Results:

The results of the immunogenicity study in mice, e.g. the humoral immune responses can be seen in FIG. 20 (FIG. 20a : anti-RSV F IgG, FIG. 20b : anti-RSV F IgG2a). In general, all groups induced humoral immune responses without significant differences between F-del alone and the F-del+ T-cell antigen groups. The addition of the T-cell antigens M, P, N, and M2-1 to the mRNA vaccine encoding RSV F-del did not influence or disturb the immune response against RSV F. These findings indicate that the co-expression of T cell antigens does not affect antibody responses induced by RSV F. The immune responses could be boosted by the second immunization (see responses on day 49). In general, the IgG2a titers are 10 times higher than the IgG1 titers, indicating a predominant Th1 response. A Th1-biased immune response is considered to be an important prerequisite for a potential RSV vaccine, as Th2-biased responses have been associated with enhanced respiratory disease (ERD) in animal models.

As shown in FIG. 21a and FIG. 21b specific antigen IgGs were detected in sera of immunized mice indicating that the applied mRNA constructs are suitable to induce specific humoral immune responses. Furthermore, the results reveal that the induction of antigen-specific antibody responses for N and M2-1 can already be detected at day 28, while for M and P immunization a boost vaccination is necessary. The decreased antigen-specific antibody responses after RSV F co-immunization are likely due to different immunization doses (5 μg vs, 2.5 μg).

As can be seen from FIG. 22, all LNP-formulated mRNA vaccines (F, M+F, P+F, N+F, M2-1+F) induced the formation of RSV specific functional antibodies in mice as shown by high virus neutralizing antibody titers. The serum neutralizing antibody titer assays against RSV/A2 revealed no significant differences between the immunization groups.

As can be seen from FIG. 23 (FIG. 23a lung ICS CD8, FIG. 23b , spleen ICS CD8, FIG. 23c spleen ICS CD4), all vaccines containing RSV F, RSV M2-1, or both surprisingly induced a tissue resident memory T cell (T_(RM)) response in the lung upon intramuscular immunization, as an increase of CD8⁺ T_(RM) cells specific for F or especially for M2-1 could be observed. The induction of a T_(RM) cell response in the lung might be favorable for a potential RSV vaccine, since lung T_(RM) cells (CD69⁺ and CD103⁺) are proposed to protect against RSV infection. The splenocyte analysis revealed that an immunization with RSV F and especially RSV M2-1 lead to an increase of antigen-specific CD8⁺ and CD4⁺ T cells secreting IFNγ and TNF indicating the induction of a systemic T cell response in addition to a site-specific response.

To conclude, the results show that RSV M2-1 induces the most promising T cell immune responses without affecting antibody responses induced by RSV F. Thus, a vaccine containing mRNAs encoding for RSV F in combination with RSV M2-1 might increase the protective efficacy against RSV infection.

Example 13: Clinical Development of a RSV mRNA Vaccine Composition

To demonstrate safety and efficiency of the RSV mRNA vaccine composition, a clinical trial (phase 1) is initiated. For clinical development, RNA is used that has been produced under GMP conditions (e.g. using a procedure as described in WO2016/180430).

In the clinical trial, a cohort of healthy human volunteers is intramuscularly injected with respective LNP formulated vaccine compositions comprising favorable UTR combinations.

In order to assess the safety profile of the vaccine compositions according to the invention, subjects are monitored after administration (vital signals, vaccination site tolerability assessments, hematologic analysis).

The efficacy of the immunization is analyzed by determination of virus neutralizing titers (VNT) in sera from vaccinated subjects. Blood samples are collected on day 0 as baseline and after completed vaccination. Sera are analyzed for virus neutralizing antibodies. 

1. An artificial RNA comprising a) at least one heterologous 5′ untranslated region (5′-UTR) and/or at least one heterologous 3′ untranslated region (3′-UTR); and b) at least one coding sequence operably linked to said 3′-UTR and/or 5′-UTR encoding at least one antigenic peptide or protein derived from a RSV fusion (F) protein or a fragment or variant thereof.
 2. Artificial RNA according to claim 1, wherein the at least one heterologous 3′-UTR comprises a nucleic acid sequence derived from a 3′-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1 and RPS9, or from a homolog, a fragment or a variant of any one of these genes.
 3. Artificial RNA according to claim 1 or 2, wherein the at least one heterologous 5′-UTR comprises a nucleic acid sequence derived from a 5′-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2, or from a homolog, a fragment or variant of any one of these genes.
 4. Artificial RNA according to any one of the preceding claims, comprising a-1. at least one 5′-UTR derived from a 5′-UTR of a HSD174 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or a-2. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or a-3. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or a-4. at least one 5′-UTR from a 5′-UTR of a NOSIP gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or a-5. at least one 5′-UTR derived from a 5′-UTR of a MP68 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or b-1. at least one 5′-UTR derived from a 5′-UTR of a UBQLN2 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or b-2. at least one 5′-UTR derived from a 5′-UTR of a ASAH1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or b-3. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or b-4. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or b-5. at least one 5′-UTR derived from a 5′-UTR of a NOSIP gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3T-UTR derived from a 3′-UTR of a COX6B1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or c-1. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or c-2. at least one 5′-UTR derived from a 5′-UTR of a NOSIP gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or c-3. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or c-4. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or c-5. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or d-1. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a PSMB3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or d-2. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or d-3. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a GNAS gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or d-4. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or d-5. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or e-1. at least one 5′-UTR derived from a 5′-UTR of a TUBB4B gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or e-2. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or e-3. at least one 5′-UTR derived from a 5′-UTR of a MP68 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or e-4. at least one 5′-UTR derived from a 5′-UTR of a NOSIP gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or e-5. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or e-6. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or f-1. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a GNAS gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or f-2. at least one 5′-UTR derived from a 5′-UTR of a ATP5A1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or f-3. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3-UTR of a COX6B1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or f-4. at least one 5′-UTR derived from a 5′-UTR of a HSD17B4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3-UTR derived from a 3′-UTR of a GNAS gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or f-5. at least one 5′-UTR derived from a 5′-UTR of a MP68 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1 ene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or g-1. at least one 5′-UTR derived from a 5′-UTR of a MP68 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3-UTR derived from a 3′-UTR of a NDUFA1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or g-2. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or g-3. at least one 5′-UTR derived from a 5′-UTR of a NDUFA4 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a GNAS gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or g-4. at least one 5′-UTR derived from a 5′-UTR of a NOSIP gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or g-5. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or h-1. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or h-2. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a GNAS gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or h-3. at least one 5′-UTR derived from a 5′-UTR of a RPL31 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a NDUFA1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or h-4. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a CASP1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or h-5. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a COX6B1 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof; or i-1. at least one 5′-UTR derived from a 5′-UTR of a SLC7A3 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a RPS9 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof. i-2, at least one 5′-UTR derived from a 5′-UTR of a RPL32 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof and at least one 3′-UTR derived from a 3′-UTR of a ALB7 gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof. i-3. at least one 3′-UTR derived from a 3′-UTR of a alpha-globin gene gene, or from a corresponding RNA sequence, homolog, fragment or variant thereof.
 5. Artificial RNA according to claim 4 comprising UTR elements according to a-1 (HSD17B4/PSMB3), a-4 (NDUFA4/PSMB3), c-1 (NDUFA4/RPS9), e-4 (NOSIP/RPS9), g-2 (NDUFA4/CASP1), i-2 (RPL32/ALB7), or i-3 (alpha-globin), preferably a-1 (HSD17B4/PSMB3)
 6. Artificial RNA according to any one of the preceding claims, wherein said 5′-UTR derived from a HSD17B4 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1 or 2 or a fragment or a variant thereof; said 5′-UTR derived from a ASAH1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or 4 or a fragment or a variant thereof; said 5′-UTR derived from a ATP5A1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5 or 6 or a fragment or a variant thereof; said 5′-UTR derived from a MP68 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7 or 8 or a fragment or a variant thereof; said 5′-UTR derived from a NDUFA4 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9 or 10 or a fragment or a variant thereof; said 5′-UTR derived from a NOSIP gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11 or 12 or a fragment or a variant thereof; said 5′-UTR derived from a RPL31 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or 14 or a fragment or a variant thereof; said 5′-UTR derived from a RPL32 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21 or 22 or a fragment or a variant thereof; said 5′-UTR derived from a SLC7A3 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or 16 or a fragment or a variant thereof; said 5′-UTR derived from a TUBB48 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17 or 18 or a fragment or a variant thereof; said 5′-UTR derived from a UBQLN2 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19 or 20 or a fragment or a variant thereof; said 3′-UTR derived from a PSMB3 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23 or 24 or a fragment or a variant thereof; said 3′-UTR derived from a CASP1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25 or 26 or a fragment or a variant thereof; said 3′-UTR derived from a COX6B1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27 or 28 or a fragment or a variant thereof; said 3′-UTR derived from a GNAS gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29 or 30 or a fragment or a variant thereof; said 3′-UTR derived from a NDUFA1 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31 or 32 or a fragment or a variant thereof; said 3′-UTR derived from a RPS9 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33 or 34 or a fragment or a variant thereof; 1 said 3′-UTR derived from a ALB7 gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35 or 36 or a fragment or a variant thereof; said 3′-UTR derived from a alpha-globin gene comprises or consists of a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37 or 38 or a fragment or a variant thereof.
 7. Artificial RNA according to any one of the preceding claims, wherein the at least one antigenic peptide or protein derived from RSV F protein is a full-length F protein (F0) or an F protein with deleted C-terminus (F-del), or a fragment or a variant thereof.
 8. Artificial RNA according to any one of the preceding claims, wherein the RSV F protein is designed to stabilize the antigen in pre-fusion conformation.
 9. Artificial RNA according to any one of the preceding claims, wherein the RSV F protein comprises a DSCav1 mutation (S155C. S290C, S190F, and V207L), or a fragment or a variant thereof.
 10. Artificial RNA according to any one of the preceding claims, wherein the RSV F protein comprises the two subunits F2 and F1 in a single polypeptide chain, wherein F2 and F1 are connected via a linker element, preferably a GS linker, to generate a stable F2-linker-F1 protein, wherein said F2-linker-F1 protein preferably lacks aa104-aa144.
 11. Artificial RNA according to any one of the preceding claims, wherein the RSV F protein comprises at least one further mutation selected from (S46G, A149C, S215P, Y458C, K465Q), (S46G, E92D, A149C, S215P, Y458C, K465Q), (S46G, N671, E92D, A149C, S215P, Y458C, K465Q), (A149C, Y458C), (N183GC, N428C), (Q98C, Q361C, S46G, E92D, L95M, S215P, I217P, I221M, R429K, K465Q), (Q98C, Q361C, L95M, I221M, R429K), or (N183GC, N428C, S46G, N671, E92D, S215P, K465Q) or a fragment or a variant thereof.
 12. Artificial RNA according to any one of the preceding claims, wherein the RSV F protein is selected from F0, F-del, F0_DSCav1, F_DSCav1_mut0, F_DSCav1_mut1, F_DSCav1_mut2, F_DSCav1_mut3, F_DSCav1_mut4, F_DSCav1_mut5, F_DSCav1_mut6, F_DSCav1_mut7, F_DSCav1_mut8, F-del_DSCav1, F-del_DSCav1_mut0, F-del_DSCav1_mut1, F-del_DSCav1_mut2, F-del_DSCav1_mut3, F-del_DSCav1_mut4, F-del_DSCav1_mut5, F-del_DSCav1_mut6, F-del_DSCav1_mut7, F-del_DSCav1_mut8 or a fragment or a variant thereof.
 13. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 68, 483, 898, 1267, 1636, 2005, 2374, 2743, 3112, 3481, 3850, 4219, 4588, 4957, 5326, 5695, 6064, 6433, 6802, 7171, 7540, 7909, 8279-9683, 11726, 12095, 12464, 12833, 13940, 14309, 14678, 15047, 15416, 15785, 13202, 13571, 16154, 16523, 16892, 17261, 17630, 17999, 18368, 18737, 19106, 19475 or a fragment or variant of any of these sequences.
 14. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 69-77, 484-492, 899-906, 1268-1275, 1637-1644, 2006-2013, 2375-2382, 2744-2751, 3113-3120, 3482-3489, 3851-3858, 4220-4227, 4589-4596, 4958-4965, 5327-5334, 5696-5703, 6065-6072, 6434-6441, 6803-6810, 7172-7179, 7541-7548, 7910-7917, 11727-11734, 12096-12103, 12465-12472, 12834-12841, 13941-13948, 14310-14317, 14679-14686, 15048-15055, 15417-15424, 15786-15793, 13203-13210, 13572-13579, 16155-16162, 16524-16531, 16893-16900, 17262-17269, 17631-17638, 18000-18007, 18369-18376, 18738-18745, 19107-19114, 19476-19483, 21363-21384, 21389-21410 or a fragment or a fragment or variant of any of these sequences.
 15. Artificial RNA according to any one of the preceding claims, wherein the at least one coding sequence is a codon modified coding sequence, wherein the amino acid sequence encoded by the at least one codon modified coding sequence is preferably not being modified compared to the amino acid sequence encoded by the corresponding wild type coding sequence.
 16. Artificial RNA according to claim 15, wherein the at least one codon modified coding sequence is selected from C maximized coding sequence, CAI maximized coding sequence, human codon usage adapted coding sequence, G/C content modified coding sequence, and G/C optimized coding sequence, or any combination thereof.
 17. Artificial RNA according to claim 15 or 16, wherein the at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one SEQ ID NOs: 70-77, 485-492, 899-906, 1268-1275, 1637-1644, 2006-2013, 2375-2382, 2744-2751, 3113-3120, 3482-3489, 3851-3858, 4220-4227, 4589-4596, 4958-4965, 5327-5334, 5696-5703, 6065-6072, 6434-6441, 6803-6810, 7172-7179, 7541-7548, 7910-7917, 11728-11734, 12097-12103, 12465-12472, 12834-12841, 13941-13948, 14310-14317, 14679-14686, 15048-15055, 15417-15424, 15786-15793, 13203-13210, 13572-13579, 16155-16162, 16524-16531, 16893-16900, 17262-17269, 17631-17638, 18000-18007, 18369-18376, 18738-18745, 19107-19114, 19476-19483, 21363-21384, 21389-21410 or a fragment or variant of any of these sequences.
 18. Artificial RNA according to claim 15 to 17, wherein the at least one coding sequence comprises a codon modified coding sequence comprising a nucleic acid sequence being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 70-71, 75-77, 485-486, 490-492, 899-900, 904-906, 1268-1269, 1273-1275, 1637-1638, 1642-1644, 2006-2007, 2011-2013, 2375-2376, 2380-2382, 2744-2745, 2749-2751, 3113-3114, 3118-3120, 3482-3483, 3487-3489, 3852, 4221, 4590, 4959, 5328, 5697, 6066, 6435, 6804, 7173, 7542, 7911, 3856-3858, 4225-4227, 4594-4596, 4963-4965, 5332-5334, 5701-5703, 6070-6072, 6439-6441, 6808-6810, 7177-7179, 7546-7548, 7915-7917, 11728, 11732-11734, 12097, 12101-12103, 12465, 12466, 12470-12472, 12834, 12835, 12839-12841, 13941, 13942, 13946-13948, 14310, 14311, 14315-14317, 14679, 14680, 14684-14686, 15048, 15049, 15053-15055, 15417, 15418, 15422-15424, 15786, 15787, 15791-15793, 13203, 13204, 13208-13210, 13572, 13573, 13577-13579, 16155, 16156, 16160-16162, 16524, 16525, 16529-16531, 16893, 16894, 16898-16900, 17262, 17263, 17267-17269, 17631, 17632, 17636-17638, 18000, 18001, 18005-18007, 18369, 18370, 18374-18376, 18738, 18739, 18743-18745, 19107, 19108, 19112-19114, 19476, 19477, 19481-19483, 21363-21384, 21389-21410 or a fragment or variant of any of these sequences.
 19. Artificial RNA according to any one of the preceding claims, wherein the RNA is an mRNA, a self-replicating RNA, a circular RNA, or a replicon RNA.
 20. Artificial RNA according to claim 19, wherein the RNA is an mRNA.
 21. Artificial RNA according to any one of the preceding claims, wherein the RNA comprises a 5′-cap structure, preferably m7G, cap0, cap1, cap2, a modified cap0 or a modified cap1 structure.
 22. Artificial RNA according to any one of the preceding claims, wherein the RNA comprises at least one poly(A) sequence, preferably comprising 30 to 150 adenosine nucleotides, more preferably comprising 100 adenosine nucleotides and/or at least one poly(C) sequence, preferably comprising 10 to 40 cytosine nucleotides.
 23. Artificial RNA according to any one of the preceding claims, wherein the RNA comprises at least one histone stem-loop, wherein the histone stem-loop preferably comprises a nucleic acid sequence according to SEQ ID NO: 39 or 40 or a fragment or variant thereof.
 24. Artificial RNA according to any one of the preceding claims comprising the following elements, preferably in 5′- to 3′-direction: a) 5′-cap structure, preferably as specified herein; b) optionally, 5′-UTR as specified herein, preferably at least one selected from SEQ ID NOs: 1-22; c) a ribosome binding site, preferably as specified herein d) at least one coding sequence as specified herein, preferably as specified in Table 3 and Table 4; d) 3′-UTR as specified herein, preferably at least one selected from SEQ ID NOs: 23-38; e) optionally, a poly(A) sequence, preferably as specified herein; f) optionally, a poly(C) sequence, preferably as specified herein; g) optionally, a histone stem-loop, preferably as specified herein; h) optionally, a 3′-terminal sequence element as specified herein, preferably according to according to SEQ ID NOs: 44-63, or 21322-21328;and wherein optionally at least one or more than one, preferably wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.
 25. Artificial RNA according claims 1 to 24 comprising the following elements, preferably in 5′- to 3-direction: a) 5′-cap structure, preferably as defined in claim 21, most preferably a Cap structure; b) 5′-UTR and/or 3′-UTR according to a-1, a-4, c-1, e-4, g-2, i-2, or i-3; c) at least one coding sequence as defined by any one of claims 13 to 18 or encoding a protein as defined by any one of claims 7 to 12 or; d) optionally, a poly(A) sequence, preferably as defined by claim 22; e) optionally, poly(C) sequence, preferably as defined by claim 22; f) optionally, histone stem-loop, preferably as defined by any one of claim 23; g) optionally, a 3′-terminal sequence according to SEQ ID NOs: 44-63, 21322-21328 and wherein optionally at least one or more than one, preferably wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.
 26. Artificial RNA according claims 1 to 25 comprising the following elements in 5′- to 3′-direction: a) 5′-cap structure, preferably as defined in claim 21, most preferably a Cap1 structure; b) 5′-UTR and/or 3′-UTR according to a-1, a-4, c-1, e-4, g-2, i-2, or i-3, preferably a-1; c) at least one coding sequence as defined by any one of claims 13 to 18 or encoding a protein as defined by any one of claims 7 to 12, wherein said coding sequence is located between said 5′-UTR and said 3′-UTR, preferably downstream of said 5′-UTR and upstream of said 3′-UTR; d) a histone stem-loop, preferably as defined by any one of claim 23 e) a poly(A) sequence, preferably as defined by claim 22 f) a 3′-terminal sequence according to SEQ ID NOs: 21322-21328. wherein optionally at least one or more than one, preferably wherein all uracil nucleotides are replaced by pseudouridine (ψ) nucleotides or N1-methylpseudouridine (m1ψ) nucleotides.
 27. Artificial RNA any one of the preceding claims, wherein the artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698 or a fragment or variant of any of these sequences.
 28. Artificial RNA according to claim 27, wherein said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 78-482, 11735-12094, 21415-21417, 21561-21563, 21489, 21490, 21635, 21636 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 493-897, 12104-12463, 21418-21420, 21564-21566, 21491, 21492, 21637, 21638 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 907-1266, 12473-12832, 21421-21423, 21567-21569, 21493-21495, 21639-21641 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1276-1635, 8278, 12842-13201, 21424-21426, 21570-21572, 21496-21498, 21642-21644 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1645-2004, 13949-14308, 21433-21435, 21579-21581, 21505-21507, 21651-21653 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2014-2373, 14318-14677, 21436-21438, 21582-21584, 21508-21510, 21654-21656 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2383-2742, 14687-15046, 21439-21441, 21585-21587, 21511-21513, 21657-21659 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2752-3111, 15056-15415, 21442-21444, 21588-21590, 21514-21516, 21660-21662 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3121-3480, 15425-15784, 21445-21447, 21591-21593, 21517-21519, 21663-21665 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3490-3849, 15794-16153, 1448-21450, 21594-21596, 21520-21522, 21666-21668 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3859-4218, 13211-13570, 21427-21429, 21573-21575, 21499-21501, 21645-21647 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4228-4587, 13580-13939, 21430-21432, 21576-21578, 21502-21504, 21648-21650 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4597-4956, 16163-16522, 21451-21453, 21597-21599, 21523-21525, 21669-21671 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4966-5325, 16532-16891, 21454-21456, 21600-21602, 21526-21528, 21672-21674 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5335-5694, 16901-17260, 21457-21459, 21603-21605, 21529-21531, 21675-21677 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5704-6063, 17270-17629, 21460-21462, 21606-21608, 21532-21534, 21678-21680 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6073-6432, 17639-17998, 21463-21465, 21609-21611, 21535-21537, 21681-21683 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6442-6801, 18008-18367, 21466-21468, 21612-21614, 21538-21540, 21684-21686 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6811-7170, 18377-18736, 21469-21471, 21615-21617, 21541-21543, 21687-21689 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7180-7539, 18746-19105, 21472-21474, 21618-21620, 21544-21546, 21690-21692 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7549-7908, 19115-19474, 21475-21477, 21621-21623, 21547-21549, 21693-21695 or a fragment or variant of any of these sequences; said artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7918-8277, 19484-19843, 21478-21480, 21624-21626, 21550-21552, 21696-21698 or a fragment or variant of any of these sequences;
 29. A composition comprising at least one artificial RNA as defined in any one of claims 1 to 28, wherein the composition optionally comprises at least one pharmaceutically acceptable carrier.
 30. A composition according to claim 29 comprising at least one further artificial RNA comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV selected from matrix protein M, nucleoprotein N, M2-1 protein, and/or phosphoprotein P or combinations thereof, preferably selected from M2-1.
 31. A composition according to claim 29 or 30, comprising two further artificial RNA species each comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV selected from matrix protein M, nucleoprotein N, M2-1, and phosphoprotein P.
 32. A composition according to claim 29 or 30, comprising three further artificial RNA species each comprising at least one coding sequence encoding at least one antigenic peptide or protein derived from RSV selected from matrix protein M, nucleoprotein N, M2-1, and phosphoprotein P.
 33. A composition according to claims 30 to 32, wherein the coding sequence of the further artificial RNA encodes at least one of the amino acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 9684, 10053-10133, 10134, 10503-10636, 10637, 11006-11182, 11183, 11552-11725, 19844, 20213, 20582, 20951 or a fragment or variant of any of these sequences.
 34. A composition according to claim 30 to 33, wherein the coding sequence is operably linked to a 3′-UTR and a 5′-UTR selected from a-1, a-2, a-3, a-4, a-5, b-1, b-2, b-3, b-4, b-5, c-1, c-2, c-3, c-4, c-5, d-1, d-2, d-3, d-4, d-5, e-1, e-2, e-3, e-4, e-5, e-6, f-1, f-2, f-3, f-4, f-5, g-1, g-2, g-3, g-4, g-5, h-1, h-2, h-3, h-4, h-5, i-1, i-2, or i-3.
 35. A composition according to claim 30 to 34, wherein the coding sequence comprises at least one of the nucleic acid sequences being identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 9685-9692, 10135-10142, 10638-10645, 11184-11191, 19845-19852, 20214-20221, 20583-20590, 20952-20959, 21385-21388, 21411-21414 or a fragment or a fragment or variant of any of these sequences.
 36. A composition according to claim 30 to 35, wherein the further artificial RNA comprises or consists of an RNA sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 9693-10052, 10143-10502, 10646-11005, 11192-11551, 19853-20212, 20222-20581, 20591-20950, 20960-21319, 21481-21634, 21553-21706 or a fragment or variant of any of these sequences.
 37. A composition according to claim 29 to 36 comprising an artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698 and a further artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9693-10052, 19853-20212, 21481, 21482, 21627, 21628, 21553, 21554, 21699, 21700; or an artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698 and a further artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10143-10502, 20222-20581, 21483, 21484, 21629, 21630, 21555, 21556, 21701, 21702; or an artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698 and a further artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10646-11005, 20591-20950, 21485, 21486, 21631, 21632, 21557, 21558, 21703, 21704; or an artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698 and a further artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11192-11551, 20960-21319; 21487, 21488, 21633, 21634, 21559, 21560, 21705, 21706 or an artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698 and a further artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 9693-10052, 19853-20212, 21481, 21482, 21627, 21628, 21553, 21554, 21699, 21700 and a further artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 10646-11005, 20591-20950, 21485, 21486, 21631, 21632, 21557, 21558, 21703, 21704; or an artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 78-482, 493-897, 907-1266, 1276-1635, 1645-2004, 2014-2373, 2383-2742, 2752-3111, 3121-3480, 3490-3849, 3859-4218, 4228-4587, 4597-4956, 4966-5325, 5335-5694, 5704-6063, 6073-6432, 6442-6801, 6811-7170, 7180-7539, 7549-7908, 7918-8277, 8278, 11735-12094, 12104-12463, 12473-12832, 12842-13201, 13949-14308, 14318-14677, 14687-15046, 15056-15415, 15425-15784, 15794-16153, 13211-13570, 13580-13939, 16163-16522, 16532-16891, 16901-17260, 17270-17629, 17639-17998, 18008-18367, 18377-18736, 18746-19105, 19115-19474, 19484-19843, 21415-21480, 21561-21626, 21489-21552, 21635-21698 and a further artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 9693-10052, 19853-20212, 21481, 21482, 21627, 21628, 21553, 21554, 21699, 21700 and a further artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 10646-11005, 20591-20950, 21485, 21486, 21631, 21632, 21557, 21558, 21703, 21704 and a further artificial RNA which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 10143-10502, 20222-20581, 21483, 21484, 21629, 21630, 21555, 21556, 21701,
 21702. 38. Composition according to claim 29 to 37, wherein the at least one artificial RNA and, optionally, the further artificial RNA is complexed or associated with or at least partially complexed or partially associated with one or more cationic or polycationic compound, preferably cationic or polycationic polymer, cationic or polycationic polysaccharide, cationic or polycationic lipid, cationic or polycationic protein, cationic or polycationic peptide, or any combinations thereof.
 39. Composition according to claim 38, wherein the artificial RNA and, optionally, the further artificial RNA is complexed or associated with one or more lipids, thereby forming liposomes, lipid nanoparticles, lipoplexes, and/or nanoliposomes.
 40. Composition according to claim 39, wherein the artificial RNA and, optionally, the further artificial RNA is complexed with one or more lipids thereby forming lipid nanoparticles (LNP).
 41. Composition according to claim 40, wherein the LNP comprises a cationic lipid with the formula Ill:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: L¹ or L² is each independently —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)—, —S—S—, —C(═O)S—, —SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, —NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, preferably L¹ or L² is —O(C═O)— or —(C═O)θ-; G¹ and G² are each independently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene; G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, or C₃-C₈ cycloalkenylene; R^(a) is H or C₁-C₁₂ alkyl; R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl; R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴; R⁴ is C₁-C₁₂ alkyl; R⁵ is H or C₁-C₁₂ alkyl; and x is 0, 1 or 2;
 42. Composition according to claim 41, wherein the cationic lipid is a compound of formula I, and wherein: L¹ and L² are each independently —O(C═O)— or (C═O)—O—; G³ is C₁-C₂₄ alkylene or C₁-C₂₄ alkenylene; and R³ is H or OR⁵.
 43. Composition according to any one of claims 41 to 42, wherein the cationic lipid is a compound of formula III, and wherein: L¹ and L² are each independently —O(C═O)— or (C═O)—O—; and R¹ and R² each independently have one of the following structures:


44. Composition according to any one of claims 41 to 43, wherein the cationic lipid is a compound of formula III, and wherein R³ is OH.
 45. Composition according to any one of claims 41 to 44, wherein the cationic lipid is selected from structures III-1 to III-36: No. Structure III-1

III-2

III-3

III-4

III-5

III-6

III-7

III-8

III-9

III-10

III-11

III-12

III-13

III-14

III-15

III-16

III-17

III-18

III-19

III-20

III-21

III-22

III-23

III-24

III-25

III-26

III-27

III-28

III-29

III-30

III-31

III-32

III-33

III-34

III-35

III-36


46. Composition according to any one of claims 41 to 45, wherein the cationic lipid is


47. Composition according to any one of claims 40 to 46, wherein the LNP comprises a PEG lipid with the formula (IV):

wherein R⁸ and R⁹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to
 60. 48. Composition according to claim 47, wherein in the PEG lipid R⁸ and R⁹ are saturated alkyl chains.
 49. Composition according to claim 47 or 48, wherein the PEG lipid is

wherein n has a mean value ranging from 30 to 60, preferably wherein n has a mean value of about 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, most preferably wherein n has a mean value of
 49. 50. Composition according to any one of claims 40 to 49, wherein the LNP comprises one or more neutral lipids and/or a steroid or steroid analogues.
 51. Composition according to claim 50, wherein the neutral lipid is selected from the group comprising distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE).
 52. Composition according to claim 50 or 51, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and wherein the molar ratio of the cationic lipid to DSPC is optionally in the range from about 2:1 to 8:1.
 53. Composition according to claim 50, wherein the steroid is cholesterol, and wherein the molar ratio of the cationic lipid to cholesterol is optionally in the range from about 2:1 to 1:1
 54. Composition according to any one of claims 40 to 53, wherein the LNP essentially consists of (i) at least one cationic lipid, preferably as defined in any one of claims 41 to 46; (ii) a neutral lipid, preferably as defined in any one of claims 50 to 52; (iii) a steroid or steroid analogue, preferably as defined in claim 53; and (iv) a PEG-lipid, e.g. PEG-DMG or PEG-cDMA, preferably as defined in any one of claims 47 to 49, wherein (i) to (iv) are in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid.
 55. Polypeptide encoded by the artificial RNA according to any one of claims 1 to 28, preferably having an amino acid sequence which is identical or at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 1267, 2005, 2743, 3481, 4219, 4957, 5695, 6433, 7171, 7909, 12833, 14309, 15047, 15785, 13571, 16523, 17261, 17999, 18737, 19475 or a variant of any of these polypeptides.
 56. A composition comprising the polypeptide as defined in claim 55, wherein the composition optionally comprises at least one pharmaceutically acceptable carrier or at least one artificial RNA as defined in any one of claims 1 to 28, or a composition as defined in any one of claims 29 to
 54. 57. A vaccine comprising the artificial RNA as defined in any one of claims 1 to 28, the composition as defined in any one of claims 29 to 54, the polypeptide as defined in claim 55, or the composition as defined in claim
 56. 58. Vaccine according to claim 49, wherein the artificial RNA as defined in any one of claims 1 to 28, the composition as defined in any one of claims 29 to 54, the polypeptide as defined in claim 55, or the composition as defined in claim 56 elicits an adaptive immune response.
 59. Vaccine according to claim 57 or 58, wherein the vaccine further comprises a pharmaceutically acceptable carrier and optionally at least one adjuvant.
 60. A Kit or kit of parts comprising the artificial RNA as defined in any one of claims 1 to 28, the composition as defined in any one of claims 29 to 54, the polypeptide as defined in claim 55, the composition as defined in claim 56, and/or the vaccine as defined in any one of claims 57 to 59, optionally comprising a liquid vehicle for solubilising, and optionally technical instructions providing information on administration and dosage of the components.
 61. Kit or kit of parts according to claim 60 comprising at least the following components a) at least one artificial RNA as defined in any one of claims 1 to 28 encoding at least one antigenic peptide or protein derived from a RSV fusion (F) protein, wherein said artificial RNA is preferably complexed with one or more lipids thereby forming lipid nanoparticles (LNP); and b) at least one, two or three further artificial RNA species each encoding an antigenic peptide or protein derived from RSV selected from M, N, M2-1, or P, wherein each of said further artificial RNA species are preferably complexed with one or more lipids thereby forming lipid nanoparticles (LNP), wherein components a) and b) are provided as separate entities or as a single entity.
 62. Kit or kit of parts according to claim 60 or 61 further comprising Ringer lactate solution.
 63. Artificial RNA as defined in any one of claims 1 to 28, the composition as defined in any one of claims 29 to 54, the polypeptide as defined in claim 55, the composition as defined in claim 56, the vaccine as defined in any one of claims 57 to 59, or the kit or kit of parts as defined in claim 60 to 62 for use as a medicament.
 64. Artificial RNA as defined in any one of claims 1 to 28, the composition as defined in any one of claims 29 to 54, the polypeptide as defined in claim 55, the composition as defined in claim 56, the vaccine as defined in any one of claims 57 to 59, or the kit or kit of parts as defined in claim 60 to 62 for use in the treatment or prophylaxis of an infection with a virus, preferably with RSV, or a disorder related to such an infection.
 65. A method of treating or preventing a disorder, wherein the method comprises applying or administering to a subject in need thereof the artificial RNA as defined in any one of claims 1 to 28, the composition as defined in any one of claims 29 to 54, the polypeptide as defined in claim 55, the composition as defined in claim 56, the vaccine as defined in any one of claims 57 to 59, or the kit or kit of parts as defined in claim 60 to 62
 66. Method according to claim 65, wherein the disorder is an infection with a RSV, or a disorder related to such an infection.
 67. Method according to claim 65 or 66, wherein the subject in need is a mammalian subject.
 68. Method according to claim 67, wherein the mammalian subject is a human subject, preferably a newborn, a pregnant woman, a breast-feeding woman, an elderly, and/or an immunocompromised human subject 